Cytokine receptor zcytor17

ABSTRACT

Novel polypeptides, polynucleotides encoding the polypeptides, and related compositions and methods are disclosed for zcytor17, a novel cytokine receptor. The polypeptides may be used within methods for detecting ligands that stimulate the proliferation and/or development of hematopoietic, lymphoid and myeloid cells in vitro and in vivo. Ligand-binding receptor polypeptides can also be used to block ligand activity in vitro and in vivo. The polynucleotides encoding zcytor17, are located on chromosome 5, and can be used to identify a region of the genome associated with human disease states. The present invention also includes methods for producing the protein, uses therefor and antibodies thereto.

BACKGROUND OF THE INVENTION

[0001] Hormones and polypeptide growth factors control proliferation anddifferentiation of cells of multicellular organisms. These diffusablemolecules allow cells to communicate with each other and act in concertto form cells and organs, and to repair damaged tissue. Examples ofhormones and growth factors include the steroid hormones (e.g. estrogen,testosterone), parathyroid hormone, follicle stimulating hormone, theinterleukins, platelet derived growth factor (PDGF), epidermal growthfactor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF),erythropoietin (EPO) and calcitonin.

[0002] Hormones and growth factors influence cellular metabolism bybinding to receptors. Receptors may be integral membrane proteins thatare linked to signaling pathways within the cell, such as secondmessenger systems. Other classes of receptors are soluble molecules,such as the transcription factors. Of particular interest are receptorsfor cytokines, molecules that promote the proliferation and/ordifferentiation of cells. Examples of cytokines include erythropoietin(EPO), which stimulates the development of red blood cells;thrombopoietin (TPO), which stimulates development of cells of themegakaryocyte lineage; and granulocyte-colony stimulating factor(G-CSF), which stimulates development of neutrophils. These cytokinesare useful in restoring normal blood cell levels in patients sufferingfrom anemia, thrombocytopenia, and neutropenia or receiving chemotherapyfor cancer.

[0003] The demonstrated in vivo activities of these cytokines illustratethe enormous clinical potential of, and need for, other cytokines,cytokine agonists, and cytokine antagonists. The present inventionaddresses these needs by providing new a hematopoietic cytokinereceptor, as well as related compositions and methods.

[0004] The present invention provides such polypeptides for these andother uses that should be apparent to those skilled in the art from theteachings herein. These and other aspects of the invention will becomeevident upon reference to the following detailed description of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005]FIG. 1 is a multiple alignment of zcytor17 polynucleotidesequences SEQ ID NO:46, SEQ ID NO:18, SEQ ID NO:54, SEQ ID NO:2, and SEQID NO:22.

[0006]FIG. 2 is an alignment of human zcytor17 (ZCYTOR) (SEQ ID NO:54)and mouse zcytor17 (M17R-O) (SEQ ID NO:57). Between the two seauences,identical residues (:), Conserved residues (.) and gaps (-) areindicated.

DESCRIPTION OF THE INVENTION

[0007] Within one aspect, the present invention provides an isolatedpolynucleotide that encodes a polypeptide comprising a sequence of aminoacid residues that is at least 90% identical to an amino acid sequenceselected from the group consisting of: (a) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 20 (Ala) to amino acidnumber 227 (Pro); (b) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 20 (Ala) to amino acid number 519 (Glu); (c) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 20(Ala) to amino acid number 543 (Leu); (d) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 544 (Lys) to amino acidnumber 732 (Val); (e) the amino acid sequence as shown in SEQ ID NO:46from amino acid number 544 (Lys) to amino acid number 649 (Ile); (f) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 20(Ala) to amino acid number 732 (Val); (g) the amino acid sequence asshown in SEQ ID NO:46 from amino acid number 20 (Ala) to amino acidnumber 649 (Ile); (h) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 1 (Met) to amino acid number 732 (Val); and (i)the amino acid sequence as shown in SEQ ID NO:46 from amino acid number1 (Met) to amino acid number 649 (Ile). In one embodiment, the isolatedpolynucleotide disclosed above comprises a sequence of amino acidresidues that is selected from the group consisting of: (a) the aminoacid sequence as shown in SEQ ID NO:2 from amino acid number 20 (Ala) toamino acid number 227 (Pro); (b) the amino acid sequence as shown in SEQID NO:2 from amino acid number 20 (Ala) to amino acid number 519 (Glu);(c) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 20 (Ala) to amino acid number 543 (Leu); (d) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 544 (Lys) toamino acid number 732 (Val); (e) the amino acid sequence as shown in SEQID NO:46 from amino acid number 544 (Lys) to amino acid number 649(Ile); (f) the amino acid sequence as shown in SEQ ID NO:2 from aminoacid number 20 (Ala) to amino acid number 732 (Val); (g) the amino acidsequence as shown in SEQ ID NO:46 from amino acid number 20 (Ala) toamino acid number 649 (Ile); (h) the amino acid sequence as shown in SEQID NO:2 from amino acid number 1 (Met) to amino acid number 732 (Val);and (i) the amino acid sequence as shown in SEQ ID NO:46 from amino acidnumber 1 (Met) to amino acid number 649 (Ile). In another embodiment,the isolated polynucleotide disclosed above comprises a sequenceselected from the group consisting of: (a) a polynucleotide as shown inSEQ ID NO:1 from nucleotide number 228 to amino acid number 851; (b) apolynucleotide as shown in SEQ ID NO:1 from nucleotide number 228 toamino acid number 1727; (c) a polynucleotide as shown in SEQ ID NO:1from nucleotide number 228 to amino acid number 1799; (d) apolynucleotide as shown in SEQ ID NO:1 from nucleotide number 1800 toamino acid number 2366; (e) a polynucleotide as shown in SEQ ID NO:45from nucleotide number 1791 to amino acid number 2108; (f) apolynucleotide as shown in SEQ ID NO:1 from nucleotide number 228 toamino acid number 2366; (g) a polynucleotide as shown in SEQ ID NO:45from nucleotide number 219 to amino acid number 2108; (h) apolynucleotide as shown in SEQ ID NO:1 from nucleotide number 171 toamino acid number 2366; (i) a polynucleotide as shown in SEQ ID NO:45from nucleotide number 162 to amino acid number 2108; and (j) apolynucleotide sequence complementary to (a) through (i). In anotherembodiment, the isolated polynucleotide disclosed above encodes apolypeptide that further comprises a transmembrane domain consisting ofresidues 520 (Ile) to 543 (Leu) of SEQ ID NO:2. In another embodiment,the isolated polynucleotide disclosed above encodes a polypeptide thatfurther comprises an intracellular domain consisting of residues 544(Lys) to 732 (Val) of SEQ ID NO:2 or 544 (Lys) to 649 (Ie) of SEQ IDNO:46. In another embodiment, the isolated polynucleotide disclosedabove encodes a polypeptide that has activity as measured by cellproliferation, activation of transcription of a reporter gene, orwherein the polypeptide encoded by the polynucleotide further binds toan antibody, wherein the antibody is raised to a polypeptide comprisinga sequence of amino acids from the group consisting of: (a) thepolypeptide comprising amino acid number 20 (Ala) to 227 (Pro) of SEQ IDNO:2; (b) the polypeptide comprising amino acid number 20 (Ala) to 519(Glu) of SEQ ID NO:2; (c) the polypeptide comprising amino acid number20 (Ala) to 543 (Leu) of SEQ ID NO:2; (d) the polypeptide comprisingamino acid number 544 (Lys) to 732 (Val) of SEQ ID NO:2; (e) thepolypeptide comprising amino acid number 544 (Lys) to 649 (Ile) of SEQID NO:46; (f) the polypeptide comprising amino acid number 20 (Ala) to732 (Val) of SEQ ID NO:2; (g) the polypeptide comprising amino acidnumber 20 (Ala) to 649 (Ie) of SEQ ID NO:46; (h) the polypeptidecomprising amino acid number 1 (Met) to 732 (Val) of SEQ ID NO:2; and(i) the polypeptide comprising amino acid number 1 (Met) to 649 (Ile) ofSEQ ID NO:46, and wherein the binding of the antibody to the isolatedpolypeptide is measured by a biological or biochemical assay includingradioimmunoassay, radioimmuno-precipitation, Western blot, orenzyme-linked immunosorbent assay.

[0008] Within a second aspect, the present invention provides anexpression vector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a polypeptide that is atleast 90% identical to an amino acid sequence as shown in SEQ ID NO:2from amino acid number 20 (Ala) to 732 (Val); or is at least 90%identical to an amino acid sequence as shown in SEQ ID NO:46 from aminoacid number 20 (Ala) to 649 (Ile); and a transcription terminator,wherein the promoter is operably linked to the DNA segment, and the DNAsegment is operably linked to the transcription terminator. In oneembodiment, the expression vector disclosed above further comprises asecretory signal sequence operably linked to the DNA segment.

[0009] Within a third aspect, the present invention provides a culturedcell comprising an expression vector as disclosed above, wherein thecell expresses a polypeptide encoded by the DNA segment. In anotherembodiment, the expression vector disclosed above comprises a DNAsegment that encodes a polypeptide comprising an amino acid sequence asshown in SEQ ID NO:2 from amino acid number 20 (Ala) to 227 (Pro); or asshown in SEQ ID NO:2 from amino acid number 20 (Ala) to 519 (Glu); and atranscription terminator, wherein the promoter, DNA segment, andterminator are operably linked. In another embodiment, the expressionvector disclosed above further comprises a secretory signal sequenceoperably linked to the DNA segment. In another embodiment, theexpression vector disclosed above further comprises a transmembranedomain consisting of residues 520 (Ile) to 543 (Leu) of SEQ ID NO:2. Inanother embodiment, the expression vector disclosed above furthercomprises an intracellular domain consisting of residues 544 (Lys) to732 (Val) of SEQ ID NO:2, or residues 544 (Lys) to 649 (Ile) of SEQ IDNO:46.

[0010] Within another aspect, the present invention provides a culturedcell into which has been introduced an expression vector as disclosedabove, wherein the cell expresses a soluble receptor polypeptide encodedby the DNA segment.

[0011] Within another aspect, the present invention provides a DNAconstruct encoding a fusion protein, the DNA construct comprising: afirst DNA segment encoding a polypeptide comprising a sequence of aminoacid residues selected from the group consisting of: (a) the amino acidsequence of SEQ ID NO:2 from amino acid number 1 (Met), to amino acidnumber 19 (Ala); (b) the amino acid sequence of SEQ ID NO:54 from aminoacid number 1 (Met), to amino acid number 32 (Ala); (c) the amino acidsequence of SEQ ID NO:2 from amino acid number 20 (Ala), to amino acidnumber 227 (Pro); (d) the amino acid sequence of SEQ ID NO:2 from aminoacid number 20 (Ala), to amino acid number 519 (Glu); (e) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Ala) toamino acid number 543 (Leu); (f) the amino acid sequence as shown in SEQID NO:2 from amino acid number 520 (Ile) to amino acid number 543 (Leu);(g) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 544 (Lys) to amino acid number 732 (Val); (h) the amino acidsequence as shown in SEQ ID NO:46 from amino acid number 544 (Lys) toamino acid number 649 (Ile); (i) the amino acid sequence as shown in SEQID NO:2 from amino acid number 20 (Ala) to amino acid number 732 (Val);and (j) the amino acid sequence as shown in SEQ ID NO:46 from amino acidnumber 20 (Ala) to amino acid number 649 (Ile); and at least one otherDNA segment encoding an additional polypeptide, wherein the first andother DNA segments are connected in-frame; and wherein the first andother DNA segments encode the fusion protein.

[0012] Within another aspect, the present invention provides anexpression vector comprising the following operably linked elements: atranscription promoter; a DNA construct encoding a fusion protein asdisclosed above; and a transcription terminator, wherein the promoter isoperably linked to the DNA construct, and the DNA construct is operablylinked to the transcription terminator.

[0013] Within another aspect, the present invention provides a culturedcell comprising an expression vector as disclosed above, wherein thecell expresses a polypeptide encoded by the DNA construct.

[0014] Within another aspect, the present invention provides a method ofproducing a fusion protein comprising: culturing a cell as disclosedabove; and isolating the polypeptide produced by the cell.

[0015] Within another aspect, the present invention provides an isolatedpolypeptide comprising a sequence of amino acid residues that is atleast 90% identical to an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 fromamino acid number 20 (Ala) to amino acid number 227 (Pro); (b) the aminoacid sequence as shown in SEQ ID NO:2 from amino acid number 20 (Ala) toamino acid number 519 (Glu); (c) the amino acid sequence as shown in SEQID NO:2 from amino acid number 20 (Ala) to amino acid number 543 (Leu);(d) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 544 (Lys) to amino acid number 732 (Val); (e) the amino acidsequence as shown in SEQ ID NO:46 from amino acid number 544 (Lys) toamino acid number 649 (Ile); (f) the amino acid sequence as shown in SEQID NO:2 from amino acid number 20 (Ala) to amino acid number 732 (Val);(g) the amino acid sequence as shown in SEQ ID NO:46 from amino acidnumber 20 (Ala) to amino acid number 649 (Ile); (h) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to aminoacid number 732 (Val); and (i) the amino acid sequence as shown in SEQID NO:46 from amino acid number 1 (Met) to amino acid number 649 (Ile).In one embodiment, the isolated polypeptide disclosed above comprises asequence of amino acid residues that is selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 fromamino acid number 20 (Ala) to amino acid number 227 (Pro); (b) the aminoacid sequence as shown in SEQ ID NO:2 from amino acid number 20 (Ala) toamino acid number 519 (Glu); (c) the amino acid sequence as shown in SEQID NO:2 from amino acid number 20 (Ala) to amino acid number 543 (Leu);(d) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 544 (Lys) to amino acid number 732 (Val); (e) the amino acidsequence as shown in SEQ ID NO:46 from amino acid number 544 (Lys) toamino acid number 649 (Ile); (f) the amino acid sequence as shown in SEQID NO:2 from amino acid number 20 (Ala) to amino acid number 732 (Val);(g) the amino acid sequence as shown in SEQ ID NO:46 from amino acidnumber 20 (Ala) to amino acid number 649 (Ile); (h) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to aminoacid number 732 (Val); and (i) the amino acid sequence as shown in SEQID NO:46 from amino acid number 1 (Met) to amino acid number 649 (Ile).In another embodiment, the isolated polypeptide disclosed above furthercomprises a transmembrane domain consisting of residues 520 (Ile) to 543(Leu) of SEQ ID NO:2. In another embodiment, the isolated polypeptidedisclosed above further comprises an intracellular domain consisting ofresidues 544 (Lys) to 732 (Val) of SEQ ID NO:2 or 544 (Lys) to 649 (Ile)of SEQ ID NO:46. In another embodiment, the isolated polypeptidedisclosed above has activity as measured by cell proliferation,activation of transcription of a reporter gene, or wherein thepolypeptide encoded by the polynucleotide further binds to an antibody,wherein the antibody is raised to a polypeptide comprising a sequence ofamino acids from the group consisting of: (a) the polypeptide comprisingamino acid number 20 (Ala) to 227 (Pro) of SEQ ID NO:2; (b) thepolypeptide comprising amino acid number 20 (Ala) to 519 (Glu) of SEQ IDNO:2; (c) the polypeptide comprising amino acid number 20 (Ala) to 543(Leu) of SEQ ID NO:2; (d) the polypeptide comprising amino acid number544 (Lys) to 732 (Val) of SEQ ID NO:2; (e) the polypeptide comprisingamino acid number 544 (Lys) to 649 (Ile) of SEQ ID NO:46; (f) thepolypeptide comprising amino acid number 20 (Ala) to 732 (Val) of SEQ IDNO:2; (g) the polypeptide comprising amino acid number 20 (Ala) to 649(Ile) of SEQ ID NO:46; (h) the polypeptide comprising amino acid number1 (Met) to 732 (Val) of SEQ ID NO:2; and (i) the polypeptide comprisingamino acid number 1 (Met) to 649 (Ile) of SEQ ID NO:46, and wherein thebinding of the antibody to the isolated polypeptide is measured by abiological or biochemical assay including radioimmunoassay,radioimmuno-precipitation, Western blot, or enzyme-linked immunosorbentassay.

[0016] Within another aspect, the present invention provides a method ofproducing a zcytor17 polypeptide comprising: culturing a cell asdisclosed above; and isolating the zcytor17 polypeptide produced by thecell.

[0017] Within another aspect, the present invention provides an isolatedpolypeptide comprising an amino acid segment selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 fromamino acid number 20 (Ala) to amino acid number 227 (Pro); (b) the aminoacid sequence as shown in SEQ ID NO:2 from amino acid number 20 (Ala) toamino acid number 519 (Glu); the amino acid sequence as shown in SEQ IDNO:18; the amino acid sequence as shown in SEQ ID NO:22; and (b)sequences that are at least 90% identical to (a) or (b), wherein thepolypeptide is substantially free of transmembrane and intracellulardomains ordinarily associated with hematopoietic receptors. Withinanother aspect, the present invention provides a method of producing azcytor17 polypeptide comprising: culturing a cell as disclosed above;and isolating the zcytor17 polypeptide produced by the cell. Withinanother aspect, the present invention provides a method of producing anantibody to a zcytor17 polypeptide comprising: inoculating an animalwith a polypeptide selected from the group consisting of: (a) apolypeptide consisting of 9 to 713 amino acids, wherein the polypeptidecomprises a contiguous sequence of amino acids in SEQ ID NO:2 from aminoacid number 20 (Ala), to amino acid number 732 (Val); (b) a polypeptideconsisting of 9 to 630 amino acids, wherein the polypeptide comprises acontiguous sequence of amino acids in SEQ ID NO:46 from amino acidnumber 20 (Ala), to amino acid number 649 (Ile); (c) a polypeptidecomprising amino acid number 20 (Ala) to 227 (Pro) of SEQ ID NO:2; (d) apolypeptide comprising amino acid number 20 (Ala) to 519 (Glu) of SEQ IDNO:2; (e) a polypeptide comprising amino acid number 20 (Ala) to 543(Leu) of SEQ ID NO:2; (f) a polypeptide comprising amino acid number 544(Lys) to 732 (Val) of SEQ ID NO:2;(g) a polypeptide comprising aminoacid number 544 (Lys) to 649 (Ile) of SEQ ID NO:46; (h) a polypeptidecomprising amino acid number 20 (Ala) to 732 (Val) of SEQ ID NO:2; (i) apolypeptide comprising amino acid number 20 Ala) to 649 (Ile) of SEQ IDNO:46; (j) a polypeptide comprising amino acid number 1 (Met) to 732(Val) of SEQ ID NO:2; (k) a polypeptide comprising amino acid number 1(Met) to 649 (Ile) of SEQ ID NO:46, (l) a polypeptide comprising aminoacid residues 43 through 48 of SEQ ID NO:2; (m) a polypeptide comprisingamino acid residues 157 through 162 of SEQ ID NO:2; (n) a polypeptidecomprising amino acid residues 158 through 163 of SEQ ID NO:2; (o) apolypeptide comprising amino acid residues 221 through 226 of SEQ IDNO:2; and (p) a polypeptide comprising amino acid residues 426 through431 of SEQ ID NO:2; and wherein the polypeptide elicits an immuneresponse in the animal to produce the antibody; and isolating theantibody from the animal. Within another aspect, the present inventionprovides an antibody produced by the method as disclosed above, whichspecifically binds to a zcytor17 polypeptide. In one embodiment, theantibody disclosed above is a monoclonal antibody.

[0018] Within another aspect, the present invention provides an antibodythat specifically binds to a polypeptide as disclosed above. In oneembodiment, the antibody disclosed above binds to a polypeptide of asdisclosed above.

[0019] Within another aspect, the present invention provides a method ofdetecting, in a test sample, the presence of a modulator of zcytor17protein activity, comprising: culturing a cell into which has beenintroduced an expression vector as disclosed above, wherein the cellexpresses the zcytor17 protein encoded by the DNA segment in thepresence and absence of a test sample; and comparing levels of activityof zcytor17 in the presence and absence of a test sample, by abiological or biochemical assay; and determining from the comparison,the presence of modulator of zcytor17 activity in the test sample.

[0020] Within another aspect, the present invention provides a methodfor detecting a zcytor17 receptor ligand within a test sample,comprising: contacting a test sample with a polypeptide comprising anamino acid sequence from the group consisting of: (a) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Ala) toamino acid number 227 (Pro); (b) the amino acid sequence as shown in SEQID NO:2 from amino acid number 20 (Ala) to amino acid number 519 (Glu);the amino acid sequence as shown in SEQ ID NO:18; the amino acidsequence as shown in SEQ ID NO:22; and detecting the binding of thepolypeptide to a ligand in the sample. In one embodiment is provided themethod disclosed above wherein the polypeptide is membrane bound withina cultured cell, and the detecting step comprises measuring a biologicalresponse in the cultured cell. In another embodiment is provided themethod disclosed above wherein the biological response is cellproliferation or activation of transcription of a reporter gene.

[0021] Prior to setting forth the invention in detail, it may be helpfulto the understanding thereof to define the following terms:

[0022] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification or detection of the second polypeptide or provide sites forattachment of the second polypeptide to a substrate. In principal, anypeptide or protein for which an antibody or other specific binding agentis available can be used as an affinity tag. Affinity tags include apolyhistidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

[0023] The term “allelic variant” is used herein to denote any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

[0024] The terms “amino-terminal” and “carboxyl-terminal” are usedherein to denote positions within polypeptides. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide to denote proximity or relative position. Forexample, a certain sequence positioned carboxyl-terminal to a referencesequence within a polypeptide is located proximal to the carboxylterminus of the reference sequence, but is not necessarily at thecarboxyl terminus of the complete polypeptide.

[0025] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

[0026] The term “complements of a polynucleotide molecule” is apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

[0027] The term “contig” denotes a polynucleotide that has a contiguousstretch of identical or complementary sequence to anotherpolynucleotide. Contiguous sequences are said to “overlap” a givenstretch of polynucleotide sequence either in their entirety or along apartial stretch of the polynucleotide. For example, representativecontigs to the polynucleotide sequence 5′-ATGGCTTAGCTT-3′ are5′-TAGCTTgagtct-3′ and 3′-gtcgacTACCGA-5′.

[0028] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0029] The term “expression vector” is used to denote a DNA molecule,linear or circular, that comprises a segment encoding a polypeptide ofinterest operably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

[0030] The term “isolated”, when applied to a polynucleotide, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985).

[0031] An “isolated” polypeptide or protein is a polypeptide or proteinthat is found in a condition other than its native environment, such asapart from blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers, multimers, or alternativelyglycosylated or derivatized forms.

[0032] The term “operably linked”, when referring to DNA segments,indicates that the segments are arranged so that they function inconcert for their intended purposes, e.g., transcription initiates inthe promoter and proceeds through the coding segment to the terminator.

[0033] The term “ortholog” denotes a polypeptide or protein obtainedfrom one species that is the functional counterpart of a polypeptide orprotein from a different species. Sequence differences among orthologsare the result of speciation.

[0034] “Paralogs” are distinct but structurally related proteins made byan organism. Paralogs are believed to arise through gene duplication.For example, α-globin, globin, β-globin, and myoglobin are paralogs ofeach other.

[0035] A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

[0036] A “polypeptide” is a polymer of amino acid residues joined bypeptide bonds, whether produced naturally or synthetically. Polypeptidesof less than about 10 amino acid residues are commonly referred to as“peptides”.

[0037] “Probes and/or primers” as used herein can be RNA or DNA. DNA canbe either cDNA or genomic DNA. Polynucleotide probes and primers aresingle or double-stranded DNA or RNA, generally syntheticoligonucleotides, but may be generated from cloned cDNA or genomicsequences or its complements. Analytical probes will generally be atleast 20 nucleotides in length, although somewhat shorter probes (14-17nucleotides) can be used. PCR primers are at least 5 nucleotides inlength, preferably 15 or more nt, more preferably 20-30 nt. Shortpolynucleotides can be used when a small region of the gene is targetedfor analysis. For gross analysis of genes, a polynucleotide probe maycomprise an entire exon or more. Probes can be labeled to provide adetectable signal, such as with an enzyme, biotin, a radionuclide,fluorophore, chemiluminescer, paramagnetic particle and the like, whichare commercially available from many sources, such as Molecular Probes,Inc., Eugene, Oreg., and Amersham Corp., Arlington Heights, Ill., usingtechniques that are well known in the art.

[0038] The term “promoter” is used herein for its art-recognized meaningto denote a portion of a gene containing DNA sequences that provide forthe binding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

[0039] A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

[0040] The term “receptor” is used herein to denote a cell-associatedprotein, or a polypeptide subunit of such a protein, that binds to abioactive molecule (the “ligand”) and mediates the effect of the ligandon the cell. Binding of ligand to receptor results in a conformationalchange in the receptor (and, in some cases, receptor multimerization,i.e., association of identical or different receptor subunits) thatcauses interactions between the effector domain(s) and other molecule(s)in the cell. These interactions in turn lead to alterations in themetabolism of the cell. Metabolic events that are linked toreceptor-ligand interactions include gene transcription,phosphorylation, dephosphorylation, cell proliferation, increases incyclic AMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. Cell-surface cytokine receptors arecharacterized by a multi-domain structure as discussed in more detailbelow. These receptors are anchored in the cell membrane by atransmembrane domain characterized by a sequence of hydrophobic aminoacid residues (typically about 21-25 residues), which is commonlyflanked by positively charged residues (Lys or Arg). In general,receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g.,thyroid stimulating hormone receptor, beta-adrenergic receptor) ormultimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor,GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6receptor). The term “receptor polypeptide” is used to denote completereceptor polypeptide chains and portions thereof, including isolatedfunctional domains (e.g., ligand-binding domains).

[0041] A “secretory signal sequence” is a DNA sequence that encodes apolypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger peptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

[0042] A “soluble receptor” is a receptor polypeptide that is not boundto a cell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains.Soluble receptors can comprise additional amino acid residues, such asaffinity tags that provide for purification of the polypeptide orprovide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis. Soluble receptor polypeptides are said to be substantiallyfree of transmembrane and intracellular polypeptide segments when theylack sufficient portions of these segments to provide membrane anchoringor signal transduction, respectively.

[0043] The term “splice variant” is used herein to denote alternativeforms of RNA transcribed from a gene. Splice variation arises naturallythrough use of alternative splicing sites within a transcribed RNAmolecule, or less commonly between separately transcribed RNA molecules,and may result in several mRNAs transcribed from the same gene. Splicevariants may encode polypeptides having altered amino acid sequence. Theterm splice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

[0044] Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

[0045] All references cited herein are incorporated by reference intheir entirety.

[0046] The present invention is based in part upon the discovery of anovel DNA sequence that encodes a protein having the structure of aclass I cytokine receptor. The deduced amino acid sequence indicatedthat the encoded receptor belongs to the receptor subfamily thatincludes gp130, LIF, IL-12, oncostatin M receptor (OSM-R), WSX-1receptors (Sprecher C A et al., Biochem. Biophys. Res. Comm. 246:81-90(1998), DCRS2 (WIPO Publication No. WO00/73451), the IL-2 receptorβ-subunit and the β-common receptor (i.e., IL3, IL-5, and GM-CSFreceptor β-subunits. The polypeptide has been designated zcytor17. Thezcytor17 polynucleotide sequence encodes the entire coding sequence ofthe predicted protein. Zcytor17 is a novel cytokine receptor that may beinvolved in immune regulation, an apoptotic cellular pathway, as acell-cell signaling molecule, growth factor receptor, or extracellularmatrix associated protein with growth factor hormone activity, or thelike.

[0047] The sequence of the zcytor17 polypeptide was deduced from genomicDNA as well as identified clones that contained its correspondingpolynucleotide sequence. The clones were obtained from a prostatelibrary. Other libraries that might also be searched for such sequencesinclude PBL, testes, monocytes, thymus, spleen, lymph node, bone marrow,human erythroleukemia, lung (e.g., WI-38 cells) and acute monocyticleukemia cell lines, other lymphoid and hematopoietic cell lines, andthe like.

[0048] Nucleotide sequences of representative zcytor17-encoding DNA aredescribed in SEQ ID NO:1 (from nucleotide 171 to 2366), with its deduced732 amino acid sequence described in SEQ ID NO:2; SEQ ID NO:45 (fromnucleotide 162 to 2108), with its deduced 649 amino acid sequencedescribed in SEQ ID NO:46.; and in SEQ ID NO:53 (from nucleotide 497 to2482), with its deduced 662 amino acid sequence described in SEQ IDNO:54. In its entirety, the zcytor17 polypeptide (SEQ ID NO:2, SEQ IDNO:46 or SEQ ID NO:54) represents a full-length polypeptide segment(residue 1 (Met) to residue 732 (Val) of SEQ ID NO:2; residue 1 (Met) toresidue 649 (Ile) of SEQ ID NO:46; residue 1 (Met) to residue 662 (Ile)of SEQ ID NO:54). The domains and structural features of the zcytor17polypeptides are further described below.

[0049] Analysis of the zcytor17 polypeptide encoded by the DNA sequenceof SEQ ID NO:1 revealed an open reading frame encoding 732 amino acids(SEQ ID NO:2) comprising a predicted secretory signal peptide of 19amino acid residues (residue 1 (Met) to residue 19 (Ala) of SEQ IDNO:2), and a mature polypeptide of 713 amino acids (residue 20 (Ala) toresidue 732 (Val) of SEQ ID NO:2). Analysis of the zcytor17 polypeptideencoded by the DNA sequence of SEQ ID NO:45 revealed an open readingframe encoding 649 amino acids (SEQ ID NO:46) comprising a predictedsecretory signal peptide of 19 amino acid residues (residue 1 (Met) toresidue 19 (Ala) of SEQ ID NO:46), and a mature polypeptide of 630 aminoacids (residue 20 (Ala) to residue 649 (Ile) of SEQ ID NO:46). Analysisof the zcytor17 polypeptide encoded by the DNA sequence of SEQ ID NO:53revealed an open reading frame encoding 662 amino acids (SEQ ID NO:54)comprising a predicted secretory signal peptide of 32 amino acidresidues (residue 1 (Met) to residue 32 (Ala) of SEQ ID NO:54), and amature polypeptide of 630 amino acids (residue 33 (Ala) to residue 662(Ile) of SEQ ID NO:54). In addition to the WSXWS motif (SEQ ID NO:3)(corresponding to residues 211 to 215 of SEQ ID NO:2 and SEQ ID NO:46;and residues 224 to 228 of SEQ ID NO:54), the receptor comprises anextracellular domain (residues 20 (Ala) to 519 (Glu) of SEQ ID NO:2 andSEQ ID NO:46; residues 33 (Ala) to 532 (Glu) of SEQ ID NO:54) whichincludes a cytokine-binding domain of approximately 200 amino acidresidues (residues 20 (Ala) to 227 (Pro) of SEQ ID NO:2 and SEQ IDNO:46; residues 33 (Ala) to 240 (Pro) of SEQ ID NO:54); a domain linker(residues 122 (Thr) to 125 (Pro) of SEQ ID NO:2 and SEQ ID NO:46;residues 135 (Thr) to 138 (Pro) of SEQ ID NO:2); a penultimate strandregion (residues 194 (Phe) to 202 (Arg) of SEQ ID NO:2 and SEQ ID NO:46;residues 207 (Phe) to 215 (Arg) of SEQ ID NO:54); a fibronectin type IIIdomain (residues 228 (Cys) to 519 (Glu) of SEQ ID) NO:2 and SEQ IDNO:46; residues 241 (Cys) to 532 (Glu) of SEQ ID NO:54); a transmembranedomain (residues 520 (Ile) to 543 (Leu) of SEQ ID NO:2 and SEQ ID NO:46;residues 533 (Ile) to 556 (Leu) of SEQ ID NO:54); complete intracellularsignaling domain (residues 544 (Lys) to 732 (Val) of SEQ ID NO:2;residues 544 (Lys) to 649 (Ile) of SEQ ID NO:46; and residues 557 (Lys)to 662 (Ile) of SEQ ID NO:54) which contains a “Box I” signaling site(residues 554 (Trp) to 560 (Pro) of SEQ ID NO:2 and SEQ ID NO:46;residues 567 (Trp) to 573 (Pro) of SEQ ID NO:54), and a “Box II”signaling site (residues 617 (Gln) to 620 (Phe) of SEQ ID NO:2 and SEQID NO:46; residues 630 (Gln) to 633 (Phe) of SEQ ID NO:54). Thoseskilled in the art will recognize that these domain boundaries areapproximate, and are based on alignments with known proteins andpredictions of protein folding. In addition to these domains, conservedreceptor features in the encoded receptor include (as shown in SEQ IDNO:2 and SEQ ID NO:46) a conserved Cys residue at position 30 (position43 as shown in SEQ ID NO:54), CXW motif (wherein X is any amino acid) atpositions 40-42 (positions 53-55 as shown in SEQ ID NO:54), Trp residueat position 170 (position 183 as shown in SEQ ID NO:54), and a conservedArg residue at position 202 (position 215 as shown in SEQ ID NO:54). Thecorresponding polynucleotides encoding the zcytor17 polypeptide regions,domains, motifs, residues and sequences described above are as shown inSEQ ID NO:1, SEQ ID NO:45, and SEQ ID NO:53.

[0050] Moreover, truncated forms of the zcytor17 polypeptide appear tobe naturally expressed. Both forms encode soluble zcytor17 receptors. Apolynucleotide encoding a “long-form” of the soluble zcytor17 receptor,truncated within the fibronectin type III domain, is shown in SEQ IDNO:17 and the corresponding polypeptide is shown in SEQ ID NO:18. Thistruncated form encodes residues 1 (Met) through 324 (Lys) of SEQ ID NO:2and SEQ ID NO:46), and thus comprises an intact signal sequence, WSXWS(SEQ ID NO:3) motif, linker, cytokine binding domain, penultimatestrand, and conserved, Cys, CXW motif, Trp and Arg residues as describedabove. A polynucleotide encoding a “short-form” of the soluble zcytor17receptor, truncated at the end of the cytokine binding domain is shownin SEQ ID NO:21 and the corresponding polypeptide is shown in SEQ IDNO:22. This truncated form encodes a 239 residue polypeptide that isidentical to residues 1 (Met) through 225 (Glu) of SEQ ID NO:2 and SEQID NO:46 and then diverges, and thus comprises an intact signalsequence, WSXWS (SEQ ID NO:3) motif, linker, cytokine binding domain,penultimate strand, and conserved, Cys, CXW motif, Trp and Arg residuesas described above. A multiple alignment of the truncated forms comparedto the full-length forms of zcytor17 is shown in FIG. 1.

[0051] Moreover, the zcytor17 cDNA of SEQ ID NO:1, SEQ ID NO:45, SEQ IDNO:17, and SEQ ID NO:21 encode polypeptides that may use an alternativeinitiating methionine (at nucleotide 75 of SEQ ID NO:1, at nucleotide 66of SEQ ID NO:45, at nucleotide 66 of SEQ ID NO:17, and at nucleotide 66of SEQ ID NO:21) that would encode a polypeptide in the same openreading frame (ORF) as the zcytor17 polypeptides of SEQ ID NO:2, SEQ IDNO:46, SEQ ID NO:18, and SEQ ID NO:22. Use of the alternative initiatingmethionine would add 32 amino acids (shown in SEQ ID NO:48) in-frame tothe N-terminus of SEQ ID NO:2, SEQ ID NO:46, SEQ ID NO:18, and SEQ IDNO:2. In addition, nucleotide 536 of SEQ ID NO:53 may serve as analternative initiating methionine, thus generating the same N-terminus(starting at amino acid 14 (Met) of SEQ ID NO:54) and signal polypeptidesequence, as SEQ ID NO:2, SEQ ID NO:46, SEQ ID NO:18, and SEQ ID NO:22.Moreover, the second Met at amino acid number 2 in the SEQ ID NO:2, SEQID NO:46, SEQ ID NO:18, and SEQ ID NO:22 sequences (similarly at aminoacid number 15 (Met) in SEQ ID NO:54) may also serve as an alternativestarting methionine for the polypeptides.

[0052] The presence of transmembrane regions, and conserved and lowvariance motifs generally correlates with or defines importantstructural regions in proteins. Regions of low variance (e.g.,hydrophobic clusters) are generally present in regions of structuralimportance (Sheppard, P. et al., supra.). Such regions of low varianceoften contain rare or infrequent amino acids, such as Tryptophan. Theregions flanking and between such conserved and low variance motifs maybe more variable, but are often functionally significant because theymay relate to or define important structures and activities such asbinding domains, biological and enzymatic activity, signal transduction,cell-cell interaction, tissue localization domains and the like.

[0053] The regions of conserved amino acid residues in zcytor17,described above, can be used as tools to identify new family members.For instance, reverse transcription-polymerase chain reaction (RT-PCR)can be used to amplify sequences encoding the conserved regions from RNAobtained from a variety of tissue sources or cell lines. In particular,highly degenerate primers designed from the zcytor17 sequences areuseful for this purpose. Designing and using such degenerate primers maybe readily performed by one of skill in the art.

[0054] The present invention provides polynucleotide molecules,including DNA and RNA molecules that encode the zcytor17 polypeptidesdisclosed herein. Those skilled in the art will recognize that, in viewof the degeneracy of the genetic code, considerable sequence variationis possible among these polynucleotide molecules. SEQ ID NO:4, SEQ IDNO:47 and SEQ ID NO:55 are degenerate DNA sequences that encompass allDNAs that encode the zcytor17 polypeptide of SEQ ID NO:2, SEQ ID NO:46and SEQ ID NO:54 respectively. Those skilled in the art will recognizethat the degenerate sequences of SEQ ID NO:4, SEQ ID NO:47 and SEQ IDNO:55 also provide all RNA sequences encoding SEQ ID NO:2, SEQ ID NO:46and SEQ ID NO:54 by substituting U for T. Thus, zcytor17polypeptide-encoding polynucleotides comprising nucleotide 1 tonucleotide 2196 of SEQ ID NO:4, nucleotide 1 to nucleotide 1947 of SEQID NO:47, and nucleotide 1 to nucleotide 1986 of SEQ ID NO:55 and theirRNA equivalents are contemplated by the present invention. Table 1 setsforth the one-letter codes used within SEQ ID NO:4, SEQ ID NO:47 and SEQID NO:55 to denote degenerate nucleotide positions. “Resolutions” arethe nucleotides denoted by a code letter. “Complement” indicates thecode for the complementary nucleotide(s). For example, the code Ydenotes either C or T, and its complement R denotes A or G, A beingcomplementary to T, and G being complementary to C. TABLE 1 NucleotideResolution Complement Resolution A A T T C C G G G G C C T T A A R A|G YC|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T W A|T H A|C|TD A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T NA|C|G|T

[0055] The degenerate codons used in SEQ ID NO:4, SEQ ID NO:47 and SEQID NO:55, encompassing all possible codons for a given amino acid, areset forth in Table 2. TABLE 2 One Amino Letter Degenerate Acid CodeCodons Codon Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr TACA ACC ACG ACT ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCNGly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAAGAG GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGGCGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L CTACTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY TyrY TAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|GlnZ SAR Any X NNN

[0056] One of ordinary skill in the art will appreciate that someambiguity is introduced in determining a degenerate codon,representative of all possible codons encoding each amino acid. Forexample, the degenerate codon for serine (WSN) can, in somecircumstances, encode arginine (AGR), and the degenerate codon forarginine (MGN) can, in some circumstances, encode serine (AGY). Asimilar relationship exists between codons encoding phenylalanine andleucine. Thus, some polynucleotides encompassed by the degeneratesequence may encode variant amino acid sequences, but one of ordinaryskill in the art can easily identify such variant sequences by referenceto the amino acid sequences of SEQ ID NO:2, SEQ ID NO:46 and SEQ IDNO:54; or SEQ ID NO:57 and SEQ ID NO:93. Variant sequences can bereadily tested for functionality as described herein.

[0057] One of ordinary skill in the art will also appreciate thatdifferent species can exhibit “preferential codon usage.” In general,see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al.Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequences disclosed in SEQID NO:4, SEQ ID NO:47 and SEQ ID NO:55 serve as templates for optimizingexpression of zcytor17 polynucleotides in various cell types and speciescommonly used in the art and disclosed herein. Sequences containingpreferential codons can be tested and optimized for expression invarious species, and tested for functionality as disclosed herein.

[0058] Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO:1,SEQ ID NO:45, or SEQ ID NO:54; or SEQ ID NO:57 and SEQ ID NO:93; or asequence complementary thereto, under stringent conditions. In general,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Numerous equations for calculating T_(m) areknown in the art, and are specific for DNA, RNA and DNA-RNA hybrids andpolynucleotide probe sequences of varying length (see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(Cold Spring Harbor Press 1989); Ausubel et al., (eds.), CurrentProtocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Bergerand Kimmel (eds.), Guide to Molecular Cloning Techniques, (AcademicPress, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227(1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake,Minn.) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto,Calif.), as well as sites on the Internet, are available tools foranalyzing a given sequence and calculating T_(m) based on user definedcriteria. Such programs can also analyze a given sequence under definedconditions and identify suitable probe sequences. Typically,hybridization of longer polynucleotide sequences (e.g., >50 base pairs)is performed at temperatures of about 20-25° C. below the calculatedT_(m). For smaller probes (e.g., <50 base pairs) hybridization istypically carried out at the T_(m) or 5-10° C. below. This allows forthe maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.Higher degrees of stringency at lower temperatures can be achieved withthe addition of formamide which reduces the T_(m) of the hybrid about 1°C. for each 1% formamide in the buffer solution. Suitable stringenthybridization conditions are equivalent to about a 5 h to overnightincubation at about 42° C. in a solution comprising: about 40-50%formamide, up to about 6×SSC, about 5×Denhardt's solution, zero up toabout 10% dextran sulfate, and about 10-20 μg/ml denaturedcommercially-available carrier DNA. Generally, such stringent conditionsinclude temperatures of 20-70° C. and a hybridization buffer containingup to 6×SSC and 0-50% formamide; hybridization is then followed bywashing filters in up to about 2×SSC. For example, a suitable washstringency is equivalent to 0.1×SSC to 2×SSC, 0.1% SDS, at 55° C. to 65°C. Different degrees of stringency can be used during hybridization andwashing to achieve maximum specific binding to the target sequence.Typically, the washes following hybridization are performed atincreasing degrees of stringency to remove non-hybridized polynucleotideprobes from hybridized complexes. Stringent hybridization and washconditions depend on the length of the probe, reflected in the Tm,hybridization and wash solutions used, and are routinely determinedempirically by one of skill in the art.

[0059] As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of zcytor17 RNA. Such tissues and cells areidentified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA77:5201, 1980), and include PBLs, spleen, thymus, bone marrow, prostate,and lymph tissues, human erythroleukemia cell lines, acute monocyticleukemia cell lines, other lymphoid and hematopoietic cell lines, andthe like. Total RNA can be prepared using guanidinium isothiocyanateextraction followed by isolation by centrifugation in a CsCl gradient(Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)⁺ RNA isprepared from total RNA using the method of Aviv and Leder (Proc. Natl.Acad. Sci. USA 69:1408-12, 1972). Complementary DNA (cDNA) is preparedfrom poly(A)⁺ RNA using known methods. In the alternative, genomic DNAcan be isolated. Polynucleotides encoding zcytor17 polypeptides are thenidentified and isolated by, for example, hybridization or polymerasechain reaction (PCR) (Mullis, U.S. Pat. No. 4,683,202).

[0060] A full-length clone encoding zcytor17 can be obtained byconventional cloning procedures. Complementary DNA (cDNA) clones arepreferred, although for some applications (e.g., expression intransgenic animals) it may be preferable to use a genomic clone, or tomodify a cDNA clone to include at least one genomic intron. Methods forpreparing cDNA and genomic clones are well known and within the level ofordinary skill in the art, and include the use of the sequence disclosedherein, or parts thereof, for probing or priming a library. Expressionlibraries can be probed with antibodies to zcytor17, receptor fragments,or other specific binding partners.

[0061] The polynucleotides of the present invention can also besynthesized using DNA synthesis machines. Currently the method of choiceis the phosphoramidite method. If chemically synthesized double strandedDNA is required for an application such as the synthesis of a gene or agene fragment, then each complementary strand is made separately. Theproduction of short polynucleotides (60 to 80 bp) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. However, for producinglonger polynucleotides (>300 bp), special strategies are usuallyemployed, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length.

[0062] An alternative way to prepare a full-length gene is to synthesizea specified set of overlapping oligonucleotides (40 to 100 nucleotides).After the 3′ and 5′ short overlapping complementary regions (6 to 10nucleotides) are annealed, large gaps still remain, but the shortbase-paired regions are both long enough and stable enough to hold thestructure together. The gaps are filled and the DNA duplex is completedvia enzymatic DNA synthesis by E. coli DNA polymerase I. After theenzymatic synthesis is completed, the nicks are sealed with T4 DNAligase. Double-stranded constructs are sequentially linked to oneanother to form the entire gene sequence which is verified by DNAsequence analysis. See Glick and Pasternak, Molecular Biotechnology,Principles & Applications of Recombinant DNA, (ASM Press, Washington,D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984 andClimie et al., Proc. Natl. Acad. Sci. USA 87:633-7, 1990. Moreover,other sequences are generally added that contain signals for properinitiation and termination of transcription and translation.

[0063] The present invention further provides counterpart polypeptidesand polynucleotides from other species (orthologs). These speciesinclude, but are not limited to mammalian, avian, amphibian, reptile,fish, insect and other vertebrate and invertebrate species. Ofparticular interest are zcytor17 polypeptides from other mammalianspecies, including murine, porcine, ovine, bovine, canine, feline,equine, and other primate polypeptides. Orthologs of human zcytor17 canbe cloned using information and compositions provided by the presentinvention in combination with conventional cloning techniques. Forexample, a cDNA can be cloned using mRNA obtained from a tissue or celltype that expresses zcytor17 as disclosed herein. Suitable sources ofmRNA can be identified by probing Northern blots with probes designedfrom the sequences disclosed herein. A library is then prepared frommRNA of a positive tissue or cell line. A zcytor17-encoding cDNA canthen be isolated by a variety of methods, such as by probing with acomplete or partial human cDNA or with one or more sets of degenerateprobes based on the disclosed sequences. A cDNA can also be cloned usingPCR (Mullis, supra.), using primers designed from the representativehuman zcytor17 sequence disclosed herein. Within an additional method,the cDNA library can be used to transform or transfect host cells, andexpression of the cDNA of interest can be detected with an antibody tozcytor17 polypeptide. Similar techniques can also be applied to theisolation of genomic clones.

[0064] A polynucleotide sequence for the mouse ortholog of humanzcytor17 has been identified and is shown in SEQ ID NO:56 and thecorresponding amino acid sequence shown in SEQ ID NO:57. Analysis of themouse zcytor17 polypeptide encoded by the DNA sequence of SEQ ID NO:56revealed an open reading frame encoding 662 amino acids (SEQ ID NO:57)comprising a predicted secretory signal peptide of 45 amino acidresidues (residue 1 (Met) to residue 45 (Ala) of SEQ ID NO:57), and amature polypeptide of 617 amino acids (residue46 (Val) to residue 662(Cys) of SEQ ID NO:57). Moreover, an additional Met residue, Met (28)can be used as a starting methionine; comprising a second predictedsecretory signal peptide of 18 amino acid residues (residue 28 (Met) toresidue 45 (Ala) of SEQ ID NO:57), and the same mature polypeptide of617 amino acids (residue46 (Val) to residue 662 (Cys) of SEQ ID NO:57.In addition to the WSXWS motif (SEQ ID NO:3) corresponding to residues224-228 of SEQ ID NO:57, the receptor comprises an extracellular domainfrom residues 46 (Val) to 533 (Glu) of SEQ ID NO:57) that includes acytokine-binding domain of approximately 200 amino acid residues(residues 46 (Val) to 240 (Pro) of SEQ ID NO:57) and a fibronectin IIIdomain (residues 241 (His) to 533 (Glu) of SEQ ID NO:57); a CXW motif(residues 66 (Cys) to 68 (Trp) of SEQ ID NO:57); a domain linker(residues 142 (Thr) to 145 (Pro) of SEQ ID NO:57); a penultimate strandregion (residues 207 (Phe) to 215 (Arg) of SEQ ID NO:57); atransmembrane domain (residues 534 (Ile) to 550 (Ile) of SEQ ID NO:57);complete intracellular signaling domain (residues 551 (Lys) to 662 (Cys)of SEQ ID NO:57) which contains a “Box I” signaling site (residues 568(Cys) to 574 (Pro) of SEQ ID NO:57), and a “Box II” signaling site(residues 628 (Glu) to 631 (leu) of SEQ ID NO:57). Conserved residuescommon to class I cytokine receptors, are at residues 56 (Cys), 187(Trp), and 215 (Arg). A comparison of the human and mouse amino acidsequences reveals that both the human and orthologous polypeptidescontain corresponding structural features described above (and, see,FIG. 2). The mature sequence for the mouse zcytor17 begins at Val₄₆ (asshown in SEQ ID NO:57), which corresponds to Ala₃₃ (as shown in SEQ IDNO:54) in the human sequence. There is about 61% identity between themouse and human sequences over the entire amino acid sequencecorresponding to SEQ ID NO:54 and SEQ ID NO:57. The above percentidentity was determined using a FASTA program with ktup=1, gap openingpenalty=12, gap extension penalty=2, and substitution matrix=BLOSUM62,with other parameters set as default. The corresponding polynucleotidesencoding the mouse zcytor17 polypeptide regions, domains, motifs,residues and sequences described above are as shown in SEQ ID NO:56.

[0065] Moreover, a truncated soluble form of the mouse zcytor17 receptorpolypeptide appears to be naturally expressed. A polynucleotide sequencefor a truncated soluble form of the mouse zcytor17 receptor has beenidentified and is shown in SEQ ID NO:92 and the corresponding amino acidsequence shown in SEQ ID NO:93. Analysis of the truncated soluble mousezcytor17 polypeptide encoded by the DNA sequence of SEQ ID NO:92revealed an open reading frame encoding 547 amino acids (SEQ ID NO:93)comprising a predicted secretory signal peptide of 45 amino acidresidues (residue 1 (Met) to residue 45 (Ala) of SEQ ID NO:93), and amature polypeptide of 502 amino acids (residue46 (Val) to residue 547(Val) of SEQ ID NO:93). Moreover, an additional Met residue, Met (28)can be used as a starting methionine; comprising a second predictedsecretory signal peptide of 18 amino acid residues (residue 28 (Met) toresidue 45 (Ala) of SEQ ID NO:93), and the same mature polypeptide of502 amino acids (residue46 (Val) to residue 547 (Val) of SEQ ID NO:93.In addition to the WSXWS motif (SEQ ID NO:3) corresponding to residues224-228 of SEQ ID NO:93, the receptor comprises an extracellular domainfrom residues 46 (Val) to 533 (Trp) of SEQ ID NO:93) that includes acytokine-binding domain of approximately 200 amino acid residues(residues 46 (Val) to 240 (Pro) of SEQ ID NO:93) and a fibronectin IIIdomain (residues 241 (His) to 533 (Trp) of SEQ ID NO:93); a CXW motif(residues 66 (Cys) to 68 (Trp) of SEQ ID NO:93); a domain linker(residues 142 (Thr) to 145 (Pro) of SEQ ID NO:93); a penultimate strandregion (residues 207 (Phe) to 215 (Arg) of SEQ ID NO:93); and aC-terminal tail region (residues 534 (Leu) to 547 (Val). Conservedresidues common to class I cytokine receptors, are at residues 56 (Cys),187 (Trp), and 215 (Arg). A comparison of the human and mouse amino acidsequences, including the truncated soluble mouse zcytor17, reveals thatboth the human and orthologous polypeptides contain correspondingstructural features described above (and, see, FIG. 2). Thecorresponding polynucleotides encoding the truncated soluble mousezcytor17 polypeptide regions, domains, motifs, residues and sequencesdescribed above are as shown in SEQ ID NO:92.

[0066] Cytokine receptor subunits are characterized by a multi-domainstructure comprising an extracellular domain, a transmembrane domainthat anchors the polypeptide in the cell membrane, and an intracellulardomain. The extracellular domain may be a ligand-binding domain, and theintracellular domain may be an effector domain involved in signaltransduction, although ligand-binding and effector functions may resideon separate subunits of a multimeric receptor. The ligand-binding domainmay itself be a multi-domain structure. Multimeric receptors includehomodimers (e.g., PDGF receptor αα and ββ isoforms, erythropoietinreceptor, MPL, and G-CSF receptor), heterodimers whose subunits eachhave ligand-binding and effector domains (e.g., PDGF receptor αβisoform), and multimers having component subunits with disparatefunctions (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, and GM-CSFreceptors). Some receptor subunits are common to a plurality ofreceptors. For example, the AIC2B subunit, which cannot bind ligand onits own but includes an intracellular signal transduction domain, is acomponent of IL-3 and GM-CSF receptors. Many cytokine receptors can beplaced into one of four related families on the basis of the structureand function. Hematopoietic receptors, for example, are characterized bythe presence of a domain containing conserved cysteine residues and theWSXWS motif (SEQ ID NO:3). Cytokine receptor structure has been reviewedby Urdal, Ann. Reports Med. Chem. 26:221-228, 1991 and Cosman, Cytokine5:95-106, 1993. Under selective pressure for organisms to acquire newbiological functions, new receptor family members likely arise fromduplication of existing receptor genes leading to the existence ofmulti-gene families. Family members thus contain vestiges of theancestral gene, and these characteristic features can be exploited inthe isolation and identification of additional family members. Thus, thecytokine receptor superfamily is subdivided into several families, forexample, the immunoglobulin family (including CSF-1, MGF, IL-1, and PDGFreceptors); the hematopoietin family (including IL-2 receptor β-subunit,GM-CSF receptor α-subunit, GM-CSF receptor β-subunit; and G-CSF, EPO,IL-3, IL-4, IL-5, IL-6, IL-7, and IL-9 receptors); TNF receptor family(including TNF (p80) TNF (p60) receptors, CD27, CD30, CD40, Fas, and NGFreceptor).

[0067] Analysis of the zcytor17 sequence suggests that it is a member ofthe same receptor subfamily as the gp130, LIF, IL-12, WSX-1, IL-2receptor β-subunit, IL-3, IL-4, and IL-6 receptors. Certain receptors inthis subfamily (e.g., G-CSF) associate to form homodimers that transducea signal. Other members of the subfamily (e.g., gp130, IL-6, IL-11, andLIF receptors) combine with a second subunit (termed a β-subunit) tobind ligand and transduce a signal. Specific β-subunits associate with aplurality of specific cytokine receptor subunits. For example, theβ-subunit gp130 (Hibi et al., Cell 63:1149-1157, 1990) associates withreceptor subunits specific for IL-6, IL-11, and LIF (Gearing et al.,EMBO J. 10:2839-2848, 1991; Gearing et al., U.S. Pat. No. 5,284,755).Oncostatin M binds to a heterodimer of LIF receptor and gp130. CNTFbinds to trimeric receptors comprising CNTF receptor, LIF receptor, andgp130 subunits.

[0068] Those skilled in the art will recognize that the sequencesdisclosed in SEQ ID NO:1, SEQ ID NO:45 and SEQ ID NO:53 representalleles of human zcytor17 and that allelic variation and alternativesplicing are expected to occur. Allelic variants of this sequence can becloned by probing cDNA or genomic libraries from different individualsaccording to standard procedures. Allelic variants of the DNA sequenceshown in SEQ ID NO:1, SEQ ID NO:45 or SEQ ID NO:53, including thosecontaining silent mutations and those in which mutations result in aminoacid sequence changes, are within the scope of the present invention, asare proteins which are allelic variants of SEQ ID NO:2, SEQ ID NO:46,SEQ ID NO:54 SEQ ID NO:57 or SEQ ID NO:93. cDNAs generated fromalternatively spliced mRNAs, which retain the properties of the zcytor17polypeptide are included within the scope of the present invention, asare polypeptides encoded by such cDNAs and mRNAs. Allelic variants andsplice variants of these sequences can be cloned by probing cDNA orgenomic libraries from different individuals or tissues according tostandard procedures known in the art. For example, the short-form andlong-form soluble zcytor17 receptors described above, and in SEQ IDNO:17 and SEQ ID NO:18 or SEQ ID NO:21 and SEQ ID NO:22 can beconsidered allelic or splice variants of zcytor17.

[0069] The present invention also provides isolated zcytor17polypeptides that are substantially similar to the polypeptides of SEQID NO:2, SEQ ID NO:46 or SEQ ID NO:54 and their orthologs, e.g., SEQ IDNO:57 and SEQ ID NO:93. The term “substantially similar” is used hereinto denote polypeptides having at least 70%, more preferably at least80%, sequence identity to the sequences shown in SEQ ID NO:2, SEQ IDNO:46 or SEQ ID NO:54 or their orthologs, e.g., SEQ ID NO:57 and SEQ IDNO:93. Such polypeptides will more preferably be at least 90% identical,and most preferably 95% or more identical to SEQ ID NO:2, SEQ ID NO:46and SEQ ID NO:54 or its orthologs.) Percent sequence identity isdetermined by conventional methods. See, for example, Altschul et al.,Bull. Math. Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc.Natl. Acad. Sci. USA 89:10915-10919, 1992. Briefly, two amino acidsequences are aligned to optimize the alignment scores using a gapopening penalty of 10, a gap extension penalty of 1, and the “blosum 62”scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 3(amino acids are indicated by the standard one-letter codes). Thepercent identity is then calculated as:$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\left\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}} \right. \\{{number}\quad {of}\quad {gaps}\quad {introduced}\quad {into}\quad {the}\quad {longer}} \\\left. {{sequence}\quad {in}\quad {order}\quad {to}\quad {align}\quad {the}\quad {two}\quad {sequences}} \right\rbrack\end{matrix}} \times 100$

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

[0070] Sequence identity of polynucleotide molecules is determined bysimilar methods using a ratio as disclosed above.

[0071] Those skilled in the art appreciate that there are manyestablished algorithms available to align two amino acid sequences. The“FASTA” similarity search algorithm of Pearson and Lipman is a suitableprotein alignment method for examining the level of identity shared byan amino acid sequence disclosed herein and the amino acid sequence of aputative variant zcytor17. The FASTA algorithm is described by Pearsonand Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson,Meth. Enzymol. 183:63 (1990).

[0072] Briefly, FASTA first characterizes sequence similarity byidentifying regions shared by the query sequence (e.g., SEQ ID NO:2, SEQID NO:46, SEQ ID NO:54, SEQ ID NO:57 and SEQ ID NO:93) and a testsequence that have either the highest density of identities (if the ktupvariable is 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then rescored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62, with other parameters set as default.These parameters can be introduced into a FASTA program by modifying thescoring matrix file (“SMATRIX”), as explained in Appendix 2 of Pearson,Meth. Enzymol. 183:63 (1990).

[0073] FASTA can also be used to determine the sequence identity ofnucleic acid molecules using a ratio as disclosed above. For nucleotidesequence comparisons, the ktup value can range between one to six,preferably from three to six, most preferably three, with other FASTAprogram parameters set as default.

[0074] The BLOSUM62 table (Table 3) is an amino acid substitution matrixderived from about 2,000 local multiple alignments of protein sequencesegments, representing highly conserved regions of more than 500 groupsof related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA89:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies canbe used to define conservative amino acid substitutions that may beintroduced into the amino acid sequences of the present invention.Although it is possible to design amino acid substitutions based solelyupon chemical properties (as discussed below), the language“conservative amino acid substitution” preferably refers to asubstitution represented by a BLOSUM62 value of greater than −1. Forexample, an amino acid substitution is conservative if the substitutionis characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to thissystem, preferred conservative amino acid substitutions arecharacterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), whilemore preferred conservative amino acid substitutions are characterizedby a BLOSUM62 value of at least 2 (e.g., 2 or 3).

[0075] Variant zcytor17 polypeptides or substantially homologouszcytor17 polypeptides are characterized as having one or more amino acidsubstitutions, deletions or additions. These changes are preferably of aminor nature, that is conservative amino acid substitutions (see Table4) and other substitutions that do not significantly affect the foldingor activity of the polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or an affinity tag. The present inventionthus includes polypeptides that comprise a sequence that is at least80%, preferably at least 90%, and more preferably 95% or more identicalto the corresponding region of SEQ ID NO:2, SEQ ID NO:46, SEQ ID NO:54,SEQ ID NO:57 or SEQ ID NO:93 excluding the tags, extension, linkersequences and the like. Polypeptides comprising affinity tags canfurther comprise a proteolytic cleavage site between the zcytor17polypeptide and the affinity tag. Suitable sites include thrombincleavage sites and factor Xa cleavage sites. TABLE 4 Conservative aminoacid substitutions Basic: arginine lysine histidine Acidic: glutamicacid aspartic acid Polar: glutamine asparagine Hydrophobic: leucineisoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small:glycine alanine serine threonine methionine

[0076] The present invention further provides a variety of otherpolypeptide fusions and related multimeric proteins comprising one ormore polypeptide fusions. For example, a zcytor17 polypeptide can beprepared as a fusion to a dimerizing protein as disclosed in U.S. Pat.Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in thisregard include immunoglobulin constant region domains.Immunoglobulin-zcytor17 polypeptide fusions can be expressed ingenetically engineered cells to produce a variety of multimeric zcytor17or more moieties, such as an affinity tag for purification and atargeting domain. Polypeptide fusions can also comprise one or morecleavage sites, particularly between domains. See, Tuan et al.,Connective Tissue Research 34:1-9, 1996.

[0077] The proteins of the present invention can also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline,N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is carried outin a cell-free system comprising an E. coli S30 extract and commerciallyavailable enzymes and other reagents. Proteins are purified bychromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung etal., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci.USA 90:10145-9, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-8, 1996). Within a third method, E. coli cells are cultured inthe absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

[0078] A limited number of non-conservative amino acids, amino acidsthat are not encoded by the genetic code, non-naturally occurring aminoacids, and unnatural amino acids may be substituted for zcytor17 aminoacid residues.

[0079] Essential amino acids in the polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc.Natl. Acad. Sci. USA 88:4498-502, 1991). In the latter technique, singlealanine mutations are introduced at every residue in the molecule, andthe resultant mutant molecules are tested for biological activity (e.g.ligand binding and signal transduction) as disclosed below to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. Sites ofligand-receptor, protein-protein or other biological interaction canalso be determined by physical analysis of structure, as determined bysuch techniques as nuclear magnetic resonance, crystallography, electrondiffraction or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,Science 255:306-312, 1992; Smith et al., J. Mol. Biol. 224:899-904,1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities ofessential amino acids can also be inferred from analysis of homologieswith related receptors.

[0080] Determination of amino acid residues that are within regions ordomains that are critical to maintaining structural integrity can bedetermined. Within these regions one can determine specific residuesthat will be more or less tolerant of change and maintain the overalltertiary structure of the molecule. Methods for analyzing sequencestructure include, but are not limited to, alignment of multiplesequences with high amino acid or nucleotide identity and computeranalysis using available software (e.g., the Insight II® viewer andhomology modeling tools; MSI, San Diego, Calif.), secondary structurepropensities, binary patterns, complementary packing and buried polarinteractions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 andCordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general,when designing modifications to molecules or identifying specificfragments determination of structure will be accompanied by evaluatingactivity of modified molecules.

[0081] Amino acid sequence changes are made in zcytor17 polypeptides soas to minimize disruption of higher order structure essential tobiological activity. For example, when the zcytor17 polypeptidecomprises one or more helices, changes in amino acid residues will bemade so as not to disrupt the helix geometry and other components of themolecule where changes in conformation abate some critical function, forexample, binding of the molecule to its binding partners. The effects ofamino acid sequence changes can be predicted by, for example, computermodeling as disclosed above or determined by analysis of crystalstructure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268,1995). Other techniques that are well known in the art compare foldingof a variant protein to a standard molecule (e.g., the native protein).For example, comparison of the cysteine pattern in a variant andstandard molecules can be made. Mass spectrometry and chemicalmodification using reduction and alkylation provide methods fordetermining cysteine residues which are associated with disulfide bondsor are free of such associations (Bean et al., Anal. Biochem.201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Pattersonet al., Anal. Chem. 66:3727-3732, 1994). It is generally believed thatif a modified molecule does not have the same disulfide bonding patternas the standard molecule folding would be affected. Another well knownand accepted method for measuring folding is circular dichrosism (CD).Measuring and comparing the CD spectra generated by a modified moleculeand standard molecule is routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are also known methods for analyzing folding andstructural similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992).

[0082] A Hopp/Woods hydrophilicity profile of the zcytor17 proteinsequence as shown in SEQ ID NO:2, SEQ ID NO:46, SEQ ID NO:54, SEQ IDNO:57 and SEQ ID NO:93 can be generated (Hopp et al., Proc. Natl. Acad.Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquieret al., Protein Engineering 11:153-169, 1998). See, FIG. 1. The profileis based on a sliding six-residue window. Buried G, S, and T residuesand exposed H, Y, and W residues were ignored. For example, in zcytor17,hydrophilic regions include amino acid residues 43 through 48 of SEQ IDNO:2 and SEQ ID NO:46 (residues 56 through 61 of SEQ ID NO:54), aminoacid residues 157 through 162 of SEQ ID NO:2 and SEQ ID NO:46 (residues170 through 175 of SEQ ID NO:54), amino acid residues 158 through 163 ofSEQ ID NO:2 and SEQ ID NO:46 (residues 171 through 176 of SEQ ID NO:54),amino acid residues 221 through 226 of SEQ ID NO:2 and SEQ ID NO:46(residues 234 through 239 of SEQ ID NO:54), and amino acid residues 426through 431 of SEQ ID NO:2 and SEQ ID NO:46 (residues 439 through 444 ofSEQ ID NO:54).

[0083] Those skilled in the art will recognize that hydrophilicity orhydrophobicity will be taken into account when designing modificationsin the amino acid sequence of a zcytor17 polypeptide, so as not todisrupt the overall structural and biological profile. Of particularinterest for replacement are hydrophobic residues selected from thegroup consisting of Val, Leu and Ile or the group consisting of Met,Gly, Ser, Ala, Tyr and Trp. For example, residues tolerant ofsubstitution could include such residues as shown in SEQ ID NO:2, SEQ IDNO:46, SEQ ID NO:54, SEQ ID NO:57 and SEQ ID NO:93. However, Cysteineresidues would be relatively intolerant of substitution.

[0084] The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between class I cytokine receptor familymembers with zcytor17. Using methods such as “FASTA” analysis describedpreviously, regions of high similarity are identified within a family ofproteins and used to analyze amino acid sequence for conserved regions.An alternative approach to identifying a variant zcytor17 polynucleotideon the basis of structure is to determine whether a nucleic acidmolecule encoding a potential variant zcytor17 polynucleotide canhybridize to a nucleic acid molecule having the nucleotide sequence ofSEQ ID NO:1, SEQ ID NO:45 or SEQ ID NO:53, as discussed above.

[0085] Other methods of identifying essential amino acids in thepolypeptides of the present invention are procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081 (1989), Bass et al., Proc. NatlAcad. Sci. USA 88:4498 (1991), Coombs and Corey, “Site-DirectedMutagenesis and Protein Engineering,” in Proteins: Analysis and Design,Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In thelatter technique, single alanine mutations are introduced at everyresidue in the molecule, and the resultant mutant molecules are testedfor biological activity as disclosed below to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., J. Biol. Chem. 271:4699 (1996).

[0086] The present invention also includes functional fragments ofzcytor17 polypeptides and nucleic acid molecules encoding suchfunctional fragments. A “functional” zcytor17 or fragment thereofdefined herein is characterized by its proliferative or differentiatingactivity, by its ability to induce or inhibit specialized cellfunctions, or by its ability to bind specifically to an anti-zcytor17antibody or zcytor17 ligand (either soluble or immobilized). Moreover,functional fragments also include the signal peptide, intracellularsignaling domain, and the like. As previously described herein, zcytor17is characterized by a class I cytokine receptor structure. Thus, thepresent invention further provides fusion proteins encompassing: (a)polypeptide molecules comprising an extracellular domain,cytokine-binding domain, or intracellular domain described herein; and(b) functional fragments comprising one or more of these domains. Theother polypeptide portion of the fusion protein may be contributed byanother class I cytokine receptor, for example, gp130, LIF, IL-12,WSX-1, IL-2 receptor β-subunit and the β-common receptor (i.e., IL3,IL-5, and GM-CSF receptor β-subunits), or by a non-native and/or anunrelated secretory signal peptide that facilitates secretion of thefusion protein.

[0087] Routine deletion analyses of nucleic acid molecules can beperformed to obtain functional fragments of a nucleic acid molecule thatencodes a zcytor17 polypeptide. As an illustration, DNA molecules havingthe nucleotide sequence of SEQ ID NO:1, SEQ ID NO:45 or SEQ ID NO:53 orfragments thereof, can be digested with Bal31 nuclease to obtain aseries of nested deletions. These DNA fragments are then inserted intoexpression vectors in proper reading frame, and the expressedpolypeptides are isolated and tested for zcytor17 activity, or for theability to bind anti-zcytor17 antibodies or zcytor17 ligand. Onealternative to exonuclease digestion is to use oligonucleotide-directedmutagenesis to introduce deletions or stop codons to specify productionof a desired zcytor17 fragment. Alternatively, particular fragments of azcytor17 polynucleotide can be synthesized using the polymerase chainreaction.

[0088] Standard methods for identifying functional domains arewell-known to those of skill in the art. For example, studies on thetruncation at either or both termini of interferons have been summarizedby Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993);Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation 1, Boynton et al.,(eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol.Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995); and Meiselet al., Plant Molec. Biol. 30:1 (1996).

[0089] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/062045) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

[0090] Variants of the disclosed zcytor17 DNA and polypeptide sequencescan be generated through DNA shuffling as disclosed by Stemmer, Nature370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated byin vitro homologous recombination by random fragmentation of a parentDNA followed by reassembly using PCR, resulting in randomly introducedpoint mutations. This technique can be modified by using a family ofparent DNAs, such as allelic variants or DNAs from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

[0091] Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized zcytor17 receptor polypeptides in host cells.Preferred assays in this regard include cell proliferation assays andbiosensor-based ligand-binding assays, which are described below.Mutagenized DNA molecules that encode active receptors or portionsthereof (e.g., ligand-binding fragments, signaling domains, and thelike) can be recovered from the host cells and rapidly sequenced usingmodem equipment. These methods allow the rapid determination of theimportance of individual amino acid residues in a polypeptide ofinterest, and can be applied to polypeptides of unknown structure.

[0092] In addition, the proteins of the present invention (orpolypeptide fragments thereof) can be joined to other bioactivemolecules, particularly cytokine receptors, to provide multi-functionalmolecules. For example, one or more domains from zcytor17 solublereceptor can be joined to other cytokine soluble receptors to enhancetheir biological properties or efficiency of production.

[0093] The present invention thus provides a series of novel, hybridmolecules in which a segment comprising one or more of the domains ofzcytor17 is fused to another polypeptide. Fusion is preferably done bysplicing at the DNA level to allow expression of chimeric molecules inrecombinant production systems. The resultant molecules are then assayedfor such properties as improved solubility, improved stability,prolonged clearance half-life, improved expression and secretion levels,and pharmacodynamics. Such hybrid molecules may further compriseadditional amino acid residues (e.g. a polypeptide linker) between thecomponent proteins or polypeptides.

[0094] Using the methods discussed herein, one of ordinary skill in theart can identify and/or prepare a variety of polypeptide fragments orvariants of SEQ ID NO:2, SEQ ID NO:46, SEQ ID NO:54, SEQ ID NO:57 andSEQ ID NO:93 that retain the signal transduction or ligand bindingactivity. For example, one can make a zcytor17 “soluble receptor” bypreparing a variety of polypeptides that are substantially homologous tothe cytokine-binding domain (residues 20 (Ala) to 227 (Pro) of SEQ IDNO:2 and SEQ ID NO:46; residues 33 (Ala) to 24o (Pro) of SEQ ID NO:54),the extracellular domain (residues 20 (Ala) to 519 (Glu) of SEQ ID NO:2and SEQ ID NO:46; residues 33 (Ala) to 532 (Glu) of SEQ ID NO:2), orallelic variants or species orthologs thereof (e.g., see SEQ ID NO:57and SEQ ID NO:93 and functional fragments thereof as described herein))and retain ligand-binding activity of the wild-type zcytor17 protein.Moreover, variant zcytor17 soluble receptors such as those shown in SEQID NO:18 and SEQ ID NO:22 can be isolated. Such polypeptides may includeadditional amino acids from, for example, part or all of thetransmembrane and intracellular domains. Such polypeptides may alsoinclude additional polypeptide segments as generally disclosed hereinsuch as labels, affinity tags, and the like.

[0095] For any zcytor17 polypeptide, including variants, solublereceptors, and fusion polypeptides or proteins, one of ordinary skill inthe art can readily generate a fully degenerate polynucleotide sequenceencoding that variant using the information set forth in Tables 1 and 2above.

[0096] The zcytor17 polypeptides of the present invention, includingfull-length polypeptides, biologically active fragments, and fusionpolypeptides, can be produced in genetically engineered host cellsaccording to conventional techniques. Suitable host cells are those celltypes that can be transformed or transfected with exogenous DNA andgrown in culture, and include bacteria, fungal cells, and culturedhigher eukaryotic cells. Eukaryotic cells, particularly cultured cellsof multicellular organisms, are preferred. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocolsin Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

[0097] In general, a DNA sequence encoding a zcytor17 polypeptide isoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are described in theliterature and are available through commercial suppliers.

[0098] To direct a zcytor17 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of zcytor17, or may be derivedfrom another secreted protein (e.g., t-PA) or synthesized de novo. Thesecretory signal sequence is operably linked to the zcytor17 DNAsequence, i.e., the two sequences are joined in the correct readingframe and positioned to direct the newly synthesized polypeptide intothe secretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain secretory signal sequences may be positionedelsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S.Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

[0099] Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence derived from amino acid 1 (Met) toamino acid 19 (Ala) of SEQ ID NO:2 and SEQ ID NO:46, or wherein asecretory signal sequence derived from amino acid 1 (Met) to amino acid32 (Ala) of SEQ ID NO:54, or amino acid 1 (Met) to amino acid 45 (Ala)of SEQ ID NO:57 or SEQ ID NO:93), or amino acid 28 (Met) to residue 45(Ala) of SEQ ID NO:57 or SEQ ID NO:93), is operably linked to anotherpolypeptide using methods known in the art and disclosed herein. Thesecretory signal sequence contained in the fusion polypeptides of thepresent invention is preferably fused amino-terminally to an additionalpeptide to direct the additional peptide into the secretory pathway.Such constructs have numerous applications known in the art. Forexample, these novel secretory signal sequence fusion constructs candirect the secretion of an active component of a normally non-secretedprotein. Such fusions may be used in vivo or in vitro to direct peptidesthrough the secretory pathway.

[0100] Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-716, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Rockville, Md. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

[0101] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

[0102] Other higher eukaryotic cells can also be used as hosts,including plant cells, insect cells and avian cells. The use ofAgrobacterium rhizogenes as a vector for expressing genes in plant cellshas been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58,1987. Transformation of insect cells and production of foreignpolypeptides therein is disclosed by Guarino et al., U.S. Pat. No.5,162,222 and WIPO publication WO 94/06463. Insect cells can be infectedwith recombinant baculovirus, commonly derived from Autographacalfornica nuclear polyhedrosis virus (AcNPV). See, King, L. A. andPossee, R. D., The Baculovirus Expression System: A Laboratory Guide,London, Chapman & Hall; O'Reilly, D. R. et al., Baculovirus ExpressionVectors: A Laboratory Manual, New York, Oxford University Press., 1994;and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methodsin Molecular Biology, Totowa, N.J., Humana Press, 1995. A second methodof making recombinant zcytor17 baculovirus utilizes a transposon-basedsystem described by Luckow (Luckow, V. A, et al., J Virol 67:4566-79,1993). This system, which utilizes transfer vectors, is sold in theBac-to-Bac™ kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, pFastBac1™ (Life Technologies) containing aTn7 transposon to move the DNA encoding the zcytor17 polypeptide into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See, Hill-Perkins, M. S. and Possee, R. D., J Gen Virol71:971-6, 1990; Bonning, B. C. et al., J Gen Virol 75:1551-6, 1994; and,Chazenbalk, G. D., and Rapoport, B., J Biol Chem 270:1543-9, 1995. Inaddition, transfer vectors can include an in-frame fusion with DNAencoding an epitope tag at the C- or N-terminus of the expressedzcytor17 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer,T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Using a techniqueknown in the art, a transfer vector containing zcytor17 is transformedinto E. Coli, and screened for bacmids which contain an interrupted lacZgene indicative of recombinant baculovirus. The bacmid DNA containingthe recombinant baculovirus genome is isolated, using common techniques,and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.Recombinant virus that expresses zcytor17 is subsequently produced.Recombinant viral stocks are made by methods commonly used in the art.

[0103] The recombinant virus is used to infect host cells, typically acell line derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells.Procedures used are generally described in available laboratory manuals(King, L. A. and Possee, R. D., ibid.; O'Reilly, D. R. et al., ibid.;Richardson, C. D., ibid.). Subsequent purification of the zcytor17polypeptide from the supernatant can be achieved using methods describedherein.

[0104] Fungal cells, including yeast cells, can also be used within thepresent invention. Yeast species of particular interest in this regardinclude Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Methods for transforming S. cerevisiae cells with exogenousDNA and producing recombinant polypeptides therefrom are disclosed by,for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S.Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S.Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075.Transformed cells are selected by phenotype determined by the selectablemarker, commonly drug resistance or the ability to grow in the absenceof a particular nutrient (e.g., leucine). A preferred vector system foruse in Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

[0105] The use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed in WIPO Publications WO 97/17450, WO97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use intransforming P. methanolica will commonly be prepared asdouble-stranded, circular plasmids, which are preferably linearizedprior to transformation. For polypeptide production in P. methanolica,it is preferred that the promoter and terminator in the plasmid be thatof a P. methanolica gene, such as a P. methanolica alcohol utilizationgene (AUG1 or AUG2). Other useful promoters include those of thedihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), andcatalase (CAT) genes. To facilitate integration of the DNA into the hostchromosome, it is preferred to have the entire expression segment of theplasmid flanked at both ends by host DNA sequences. A preferredselectable marker for use in Pichia methanolica is a P. methanolica ADE2gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;EC 4.1.1.21), which allows ade2 host cells to grow in the absence ofadenine. For large-scale, industrial processes where it is desirable tominimize the use of methanol, it is preferred to use host cells in whichboth methanol utilization genes (AUG1 and AUG2) are deleted. Forproduction of secreted proteins, host cells deficient in vacuolarprotease genes (PEP4 and PRB1) are preferred. Electroporation is used tofacilitate the introduction of a plasmid containing DNA encoding apolypeptide of interest into P. methanolica cells. It is preferred totransform P. methanolica cells by electroporation using an exponentiallydecaying, pulsed electric field having a field strength of from 2.5 to4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from1 to 40 milliseconds, most preferably about 20 milliseconds.

[0106] Prokaryotic host cells, including strains of the bacteriaEscherichia coli, Bacillus and other genera are also useful host cellswithin the present invention. Techniques for transforming these hostsand expressing foreign DNA sequences cloned therein are well known inthe art (see, e.g., Sambrook et al., ibid.). When expressing a zcytor17polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

[0107] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

[0108] Within one aspect of the present invention, a zcytor17 cytokinereceptor (including transmembrane and intracellular domains) is producedby a cultured cell, and the cell is used to screen for ligands for thereceptor, including the natural ligand, as well as agonists andantagonists of the natural ligand. To summarize this approach, a cDNA orgene encoding the receptor is combined with other genetic elementsrequired for its expression (e.g., a transcription promoter), and theresulting expression vector is inserted into a host cell. Cells thatexpress the DNA and produce functional receptor are selected and usedwithin a variety of screening systems.

[0109] Mammalian cells suitable for use in expressing the novelreceptors of the present invention and transducing a receptor-mediatedsignal include cells that express a β-subunit, such as gp130, and cellsthat co-express gp130 and LIF receptor (Gearing et al., EMBO J.10:2839-2848, 1991; Gearing et al., U.S. Pat. No. 5,284,755). In thisregard it is generally preferred to employ a cell that is responsive toother cytokines that bind to receptors in the same subfamily, such asIL-6 or LIF, because such cells will contain the requisite signaltransduction pathway(s). Preferred cells of this type include BaF3 cells(Palacios and Steinmetz, Cell 41: 727-734, 1985; Mathey-Prevot et al.,Mol. Cell. Biol. 6: 4133-4135, 1986), the human TF-1 cell line (ATCCnumber CRL-2003) and the DA-1 cell line (Branch et al., Blood 69:1782,1987; Broudy et al., Blood 75:1622-1626, 1990). In the alternative,suitable host cells can be engineered to produce a β-subunit or othercellular component needed for the desired cellular response. Forexample, the murine cell line BaF3 (Palacios and Steinmetz, Cell41:727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135,1986), a baby hamster kidney (BHK) cell line, or the CTLL-2 cell line(ATCC TIB-214) can be transfected to express the mouse gp130 subunit, ormouse gp130 and LIF receptor, in addition to zcytor17. It is generallypreferred to use a host cell and receptor(s) from the same species,however this approach allows cell lines to be engineered to expressmultiple receptor subunits from any species, thereby overcomingpotential limitations arising from species specificity. In thealternative, species homologs of the human receptor cDNA can be clonedand used within cell lines from the same species, such as a mouse cDNAin the BaF3 cell line. Cell lines that are dependent upon onehematopoietic growth factor, such as IL-3, can thus be engineered tobecome dependent upon a zcytor17 ligand or anti-zcytor17 antibody.

[0110] Cells expressing functional zcytor17 are used within screeningassays. A variety of suitable assays are known in the art. These assaysare based on the detection of a biological response in the target cell.One such assay is a cell proliferation assay. Cells are cultured in thepresence or absence of a test compound, and cell proliferation isdetected by, for example, measuring incorporation of tritiated thymidineor by colorimetric assay based on the reduction or metabolic breakdownof Alymar Blue™ (AccuMed, Chicago, Ill.) or3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)(Mosman, J. Immunol. Meth. 65: 55-63, 1983). An alternative assay formatuses cells that are further engineered to express a reporter gene. Thereporter gene is linked to a promoter element that is responsive to thereceptor-linked pathway, and the assay detects activation oftranscription of the reporter gene. A preferred promoter element in thisregard is a serum response element, STAT or SRE (see, for example, Shawet al., Cell 56:563-572, 1989). A preferred such reporter gene is aluciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987).Expression of the luciferase gene is detected by luminescence usingmethods known in the art (e.g., Baumgartner et al., J. Biol. Chem.269:19094-29101, 1994; Schenborn and Goiffin, Promega Notes 41:11,1993). Luciferase assay kits are commercially available from, forexample, Promega Corp., Madison, Wis. Target cell lines of this type canbe used to screen libraries of chemicals, cell-conditioned culturemedia, fungal broths, soil samples, water samples, and the like. Forexample, a bank of cell- or tissue-conditioned media samples can beassayed on a target cell to identify cells that produce ligand. Positivecells are then used to produce a cDNA library in a mammalian cellexpression vector, which is divided into pools, transfected into hostcells, and expressed. Media samples from the transfected cells are thenassayed, with subsequent division of pools, retransfection,subculturing, and re-assay of positive cells to isolate a clonal cellline expressing the ligand. Media samples conditioned by kidney, liver,spleen, thymus, other lymphoid tissues, or T-cells are preferred sourcesof ligand for use in screening procedures.

[0111] A natural ligand for zcytor17 can also be identified bymutagenizing a cytokine-dependent cell line expressing zcytor17 andculturing it under conditions that select for autocrine growth. See WIPOpublication WO 95/21930. Within a typical procedure, cells expressingzcytor17 are mutagenized, such as with EMS. The cells are then allowedto recover in the presence of the required cytokine, then transferred toa culture medium lacking the cytokine. Surviving cells are screened forthe production of a ligand for zcytor17, such as by adding solublereceptor polypeptide comprising the zcytor17 cytokine-binding domaindescribed herein to the culture medium to compete against the ligand orby assaying conditioned media on wild-type cells compared to transfectedcells expressing the zcytor17 receptor. Preferred cell lines for usewithin this method include cells that are transfected to express gp130or gp130 in combination with LIF receptor. Preferred such host celllines include transfected CTLL-2 cells (Gillis and Smith, Nature268:154-156, 1977) and transfected BaF3 cells.

[0112] Moreover, a secretion trap method employing zcytor17 solublereceptor polypeptide can be used to isolate a zcytor17 ligand (Aldrich,et al, Cell 87: 1161-1169, 1996). A cDNA expression library preparedfrom a known or suspected ligand source is transfected into COS-7 cells.The cDNA library vector generally has an SV40 origin for amplificationin COS-7 cells, and a CMV promoter for high expression. The transfectedCOS-7 cells are grown in a monolayer and then fixed and permeabilized.Tagged or biotin-labeled zcytor17 soluble receptor, described herein, isthen placed in contact with the cell layer and allowed to bind cells inthe monolayer that express an anti-complementary molecule, i.e., azcytor17 ligand. A cell expressing a ligand will thus be bound withreceptor molecules. An anti-tag antibody (anti-Ig for Ig fusions, M2 oranti-FLAG for FLAG-tagged fusions, streptavidin, anti-Glu-Glu tag, andthe like) which is conjugated with horseradish peroxidase (HRP) is usedto visualize these cells to which the tagged or biotin-labeled zcytor17soluble receptor has bound. The HRP catalyzes deposition of a tyramidereagent, for example, tyramide-FITC. A commercially-available kit can beused for this detection (for example, Renaissance TSA-Direct™ Kit; NENLife Science Products, Boston, Mass.). Cells which express zcytor17receptor ligand will be identified under fluorescence microscopy asgreen cells and picked for subsequent cloning of the ligand usingprocedures for plasmid rescue as outlined in Aldrich, et al, supra.,followed by subsequent rounds of secretion trap assay, or conventionalscreening of cDNA library pools, until single clones are identified.

[0113] As a receptor, the activity of zcytor17 polypeptide can bemeasured by a silicon-based biosensor microphysiometer which measuresthe extracellular acidification rate or proton excretion associated withreceptor binding and subsequent physiologic cellular responses. Anexemplary device is the Cytosensor™ Microphysiometer manufactured byMolecular Devices, Sunnyvale, Calif. A variety of cellular responses,such as cell proliferation, ion transport, energy production,inflammatory response, regulatory and receptor activation, and the like,can be measured by this method. See, for example, McConnell, H. M. etal., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol.228:84-108, 1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59,1998; Van Liefde, I. Et al., Eur. J. Pharmacol. 346:87-95, 1998. Themicrophysiometer can be used for assaying eukaryotic, prokaryotic,adherent or non-adherent cells. By measuring extracellular acidificationchanges in cell media over time, the microphysiometer directly measurescellular responses to various stimuli, including agonists, ligands, orantagonists of the zcytor17 polypeptide. Preferably, themicrophysiometer is used to measure responses of a zcytor17-expressingeukaryotic cell, compared to a control eukaryotic cell that does notexpress zcytor17 polypeptide. Zcytor17-expressing eukaryotic cellscomprise cells into which zcytor17 has been transfected or infected viaadenovirus vector, and the like, as described herein, creating a cellthat is responsive to zcytor17-modulating stimuli, or are cellsnaturally expressing zcytor17, such as zcytor17-expressing cells derivedfrom lymphoid, spleen, thymus tissue or PBLs. Differences, measured byan increase or decrease in extracellular acidification, in the responseof cells expressing zcytor17, relative to a control, are a directmeasurement of zcytor17-modulated cellular responses. Moreover, suchzcytor17-modulated responses can be assayed under a variety of stimuli.Also, using the microphysiometer, there is provided a method ofidentifying agonists and antagonists of zcytor17 polypeptide, comprisingproviding cells expressing a zcytor17 polypeptide, culturing a firstportion of the cells in the absence of a test compound, culturing asecond portion of the cells in the presence of a test compound, anddetecting an increase or a decrease in a cellular response of the secondportion of the cells as compared to the first portion of the cells.Antagonists and agonists, including the natural ligand for zcytor17polypeptide, can be rapidly identified using this method.

[0114] Additional assays provided by the present invention include theuse of hybrid receptor polypeptides. These hybrid polypeptides fall intotwo general classes. Within the first class, the intracellular domain ofzcytor17, comprising approximately residues 544 (Lys) to 732 (Val) ofSEQ ID NO:2, residues 544 (Lys) to 649 (Ile) of SEQ ID NO:46, orresidues 557 (Lys) to 662 (Ile) of SEQ ID NO:54, or residues 551 (Lys)to 662 (Cys) of SEQ ID NO:57 is joined to the ligand-binding domain of asecond receptor. It is preferred that the second receptor be ahematopoietic cytokine receptor, such as mpl receptor (Souyri et al.,Cell 63:1137-1147, 1990). The hybrid receptor will further comprise atransmembrane domain, which may be derived from either receptor. A DNAconstruct encoding the hybrid receptor is then inserted into a hostcell. Cells expressing the hybrid receptor are cultured in the presenceof a ligand for the binding domain and assayed for a response. Thissystem provides a means for analyzing signal transduction mediated byzcytor17 while using readily available ligands. This system can also beused to determine if particular cell lines are capable of responding tosignals transduced by zcytor17. A second class of hybrid receptorpolypeptides comprise the extracellular (ligand-binding) domain(approximately residues 20 (Ala) to 519 (Glu) of SEQ ID NO:2 and SEQ IDNO:46; approximately residues 33 (Ala) to 532 (Glu) of SEQ ID NO:54) orcytokine-binding domain of zcytor17 (approximately residues 20 (Ala) to227 (Pro) of SEQ ID NO:2 and SEQ ID NO:46; or approximately residues 33(Ala) to 240 (Pro) of SEQ ID NO:54; approximately residues 46 (Val) to533 (Glu) of SEQ ID NO:57; or approximately residues 46 (Val) to 533(Trp) of SEQ ID NO:93) with a cytoplasmic domain of a second receptor,preferably a cytokine receptor, and a transmembrane domain. Thetransmembrane domain may be derived from either receptor. Hybridreceptors of this second class are expressed in cells known to becapable of responding to signals transduced by the second receptor.Together, these two classes of hybrid receptors enable the use of abroad spectrum of cell types within receptor-based assay systems.

[0115] Cells found to express a ligand for zcytor17 are then used toprepare a cDNA library from which the ligand-encoding cDNA may beisolated as disclosed above. The present invention thus provides, inaddition to novel receptor polypeptides, methods for cloning polypeptideligands for the receptors.

[0116] The zcytor17 structure and tissue expression suggests a role inearly hematopoietic or thymocyte development and immune responseregulation or inflammation. These processes involve stimulation of cellproliferation and differentiation in response to the binding of one ormore cytokines to their cognate receptors. In view of the tissuedistribution observed for this receptor, agonists (including the naturalligand) and antagonists have enormous potential in both in vitro and invivo applications. Compounds identified as receptor agonists are usefulfor stimulating proliferation and development of target cells in vitroand in vivo. For example, agonist compounds or antizcytor17 antibodies,are useful as components of defined cell culture media, and may be usedalone or in combination with other cytokines and hormones to replaceserum that is commonly used in cell culture. Agonists are thus useful inspecifically promoting the growth and/or development or activation ofmonocytes, T-cells, B-cells, and other cells of the lymphoid and myeloidlineages, and hematopoietic cells in culture.

[0117] Agonist ligands for zcytor17, or anti-zcytor17 antibodies andbinding partners, may be useful in stimulating cell-mediated immunityand for stimulating lymphocyte proliferation, such as in the treatmentof infections involving immunosuppression, including certain viralinfections. Additional uses include tumor suppression, where malignanttransformation results in tumor cells that are antigenic. Agonistligands or anti-zcytor17 antibodies and binding partners could be usedto induce cytotoxicity, which may be mediated through activation ofeffector cells such as T-cells, NK (natural killer) cells, or LAK(lymphoid activated killer) cells, or induced directly through apoptoticpathways. Agonist ligands, anti-zcytor17 antibodies and binding partnersmay also be useful in treating leukopenias by increasing the levels ofthe affected cell type, and for enhancing the regeneration of the T-cellrepertoire after bone marrow transplantation; or for enhancing monocyteproliferation or activation, and for diagnostic and other uses describedherein.

[0118] Antagonist ligands, compounds, or anti-zcytor17 antibodies mayfind utility in the suppression of the immune system, such as in thetreatment of autoimmune diseases, including rheumatoid arthritis,multiple sclerosis, diabetes mellitis, inflammatory bowel disease,Crohn's disease, etc. Immune suppression can also be used to reducerejection of tissue or organ transplants and grafts and to treat T-cell,B-cell or monocyte-specific leukemias or lymphomas, and other immunecell cancers, by inhibiting proliferation of the affected cell type.Moreover zcytor17 polynucleotides, anti-zcytor17 antibodies and bindingpartners can be used to detect monocytes, and aid in the diagnosis ofsuch autoimmune disease, particularly in disease states where monocytesare elevated or activated.

[0119] Zcytor17 polypeptides may also be used within diagnostic systemsfor the detection of circulating levels of ligand. Within a relatedembodiment, antibodies or other agents that specifically bind tozcytor17 receptor polypeptides can be used to detect circulatingreceptor polypeptides. Zcytor17 appears to be naturally-expressed as asoluble receptor as shown by the soluble receptor forms shown in SEQ IDNO:18 and SEQ ID NO:22. Elevated or depressed levels of ligand orreceptor polypeptides may be indicative of pathological conditions,including cancer. Soluble receptor polypeptides may contribute topathologic processes and can be an indirect marker of an underlyingdisease. For example, elevated levels of soluble IL-2 receptor in humanserum have been associated with a wide variety of inflammatory andneoplastic conditions, such as myocardial infarction, asthma, myastheniagravis, rheumatoid arthritis, acute T-cell leukemia, B-cell lymphomas,chronic lymphocytic leukemia, colon cancer, breast cancer, and ovariancancer (Heaney et al., Blood 87:847-857, 1996). Similarly, zcytor17 iselevated in activated monocytes, and hence zcytor17 and/or its solublereceptors may be associated with or serve as a marker for inflammatoryand neoplastic conditions associated therewith.

[0120] A ligand-binding polypeptide of a zcytor17 receptor, or “solublereceptor,” can be prepared by expressing a truncated DNA encoding thezcytor17 cytokine binding domain (approximately residue 20 (Ala) throughresidue 227 (Pro) of the human receptor SEQ ID NO:2 and SEQ ID NO:46;approximately residue 33 (Ala) through residue 240 (Pro) of the humanreceptor SEQ ID NO:54), or the extracellular domain (approximatelyresidue 20 (Ala) through residue 519 (Glu) of SEQ ID NO:2 and SEQ IDNO:46; approximately residue 33 (Ala) through residue 532 (Glu) of SEQID NO:54), or the corresponding region of a non-human receptor, e.g.,such as the corresponding regions described herein for SEQ ID NO:57 andSEQ ID NO:93. It is preferred that the extracellular domain be preparedin a form substantially free of transmembrane and intracellularpolypeptide segments. Moreover, ligand-binding polypeptide fragmentswithin the zcytor17 cytokine binding domain, described above, can alsoserve as zcytor17 soluble receptors for uses described herein. To directthe export of a receptor polypeptide from the host cell, the receptorDNA is linked to a second DNA segment encoding a secretory peptide, suchas a t-PA secretory peptide or a zcytor17 secretory peptide. Tofacilitate purification of the secreted receptor polypeptide, aC-terminal extension, such as a poly-histidine tag, Glu-Glu tag peptide,substance P, Flag™ peptide (Hopp et al., Bio/Technology 6:1204-1210,1988; available from Eastman Kodak Co., New Haven, Conn.) or anotherpolypeptide or protein for which an antibody or other specific bindingagent is available, can be fused to the receptor polypeptide.

[0121] In an alternative approach, a receptor extracellular domain canbe expressed as a fusion with immunoglobulin heavy chain constantregions, typically an F_(c) fragment (e.g., Fc4), which contains twoconstant region domains and lacks the variable region. Such fusions aretypically secreted as multimeric molecules wherein the Fc portions aredisulfide bonded to each other and two receptor polypeptides are arrayedin close proximity to each other. Fusions of this type can be used toaffinity purify the cognate ligand from solution, as an in vitro assaytool, to block signals in vitro by specifically titrating out ligand,and as antagonists in vivo by administering them parenterally to bindcirculating ligand and clear it from the circulation. To purify ligand,a zcytor17-Ig chimera is added to a sample containing the ligand (e.g.,cell-conditioned culture media or tissue extracts) under conditions thatfacilitate receptor-ligand binding (typically near-physiologicaltemperature, pH, and ionic strength). The chimera-ligand complex is thenseparated by the mixture using protein A, which is immobilized on asolid support (e.g., insoluble resin beads). The ligand is then elutedusing conventional chemical techniques, such as with a salt or pHgradient. In the alternative, the chimera itself can be bound to a solidsupport, with binding and elution carried out as above. Collectedfractions can be re-fractionated until the desired level of purity isreached.

[0122] Moreover, zcytor17 soluble receptors can be used as a “ligandsink,” i.e., antagonist, to bind ligand in vivo or in vitro intherapeutic or other applications where the presence of the ligand isnot desired. For example, in cancers that are expressing large amount ofbioactive zcytor17 ligand, zcytor17 soluble receptors can be used as adirect antagonist of the ligand in vivo, and may aid in reducingprogression and symptoms associated with the disease. Moreover, zcytor17soluble receptor can be used to slow the progression of cancers thatover-express zcytor17 receptors, by binding ligand in vivo that wouldotherwise enhance proliferation of those cancers. Similar in vitroapplications for a zcytor17 soluble receptor can be used, for instance,as a negative selection to select cell lines that grow in the absence ofzcytor17 ligand.

[0123] Moreover, zcytor17 soluble receptor can be used in vivo or indiagnostic applications to detect zcytor17 ligand-expressing cancers invivo or in tissue samples. For example, the zcytor17 soluble receptorcan be conjugated to a radio-label or fluorescent label as describedherein, and used to detect the presence of the ligand in a tissue sampleusing an in vitro ligand-receptor type binding assay, or fluorescentimaging assay. Moreover, a radiolabeled zcytor17 soluble receptor couldbe administered in vivo to detect ligand-expressing solid tumors througha radio-imaging method known in the art.

[0124] The molecules of the present invention have particular use in themonocyte/macrophage arm of the immune system. For example, interferongamma (IFNγ) is a potent activator of mononuclear phagocytes. Theincrease in expression of zcytor17 upon activation of THP-1 cells (ATCCNo. TIB-202) with interferon gamma suggests that this receptor isinvolved in monocyte activation. Monocytes are incompletelydifferentiated cells that migrate to various tissues where they matureand become macrophages. Macrophages play a central role in the immuneresponse by presenting antigen to lymphocytes and play a supportive roleas accessory cells to lymphocytes by secreting numerous cytokines.Macrophages can internalize extracellular molecules and upon activationhave an increased ability to kill intracellular microorganisms and tumorcells. Activated macrophages are also involved in stimulating acute orlocal inflammation. Moreover, monocyte-macrophage function has beenshown to be abnormal in a variety of diseased states. For example see,Johnston, R B, New Eng. J. Med. 318:747-752, 1998.

[0125] One of skill in the art would recognize that agonists of zcytor17are useful. For example, depressed migration of monocytes has beenreported in populations with a predisposition to infection, such asnewborn infants, patients receiving corticosteroid or otherimmunosuppressive therapy, and patients with diabetes mellitus, burns,or AIDS. Agonists for zcytor17, such as anti-zcytor17 antibodies andbinding partners, as well as the natural ligand, could result in anincrease in ability of monocytes to migrate and possibly preventinfection in these populations. There is also a profound defect ofphagocytic killing by mononuclear phagocytes from patients with chronicgranulomatous disease. This results in the formation of subcutaneousabscesses, as well as abscesses in the liver, lungs, spleen, and lymphnodes. An agonist of zcytor17 such as anti-zcytor17 antibodies andbinding partners, as well as the natural ligand, could correct orimprove this phagocytic defect. In addition, defective monocytecytotoxicity has been reported in patients with cancer andWiskott-Aldrich syndrome (eczema, thrombocytopenia, and recurrentinfections). Activation of monocytes by agonists of zcytor17 such asanti-zcytor17 antibodies and binding partners, as well as the naturalligand, could aid in treatment of these conditions. Themonocyte-macrophage system is prominently involved in severallipid-storage diseases (sphingolipidoses) such as Gaucher's disease.Resistance to infection can be impaired because of a defect inmacrophage function, which could be treated by agonists to zcytor17 suchas anti-zcytor17 antibodies and binding partners, as well as the naturalligand.

[0126] Moreover, one of skill in the art would recognize thatantagonists of zcytor17 are useful. For example, in atheroscleroticlesions, one of the first abnormalities is localization ofmonocyte/macrophages to endothelial cells. These lesions could beprevented by use of antagonists to zcytor17. Anti-zcytor17 antibodiesand binding partners can also be used as antagonists to the naturalligand of zcytor17. Moreover, monoblastic leukemia is associated with avariety of clinical abnormalities that reflect the release of thebiologic products of the macrophage, examples include high levels oflysozyme in the serum and urine and high fevers. Moreover, suchleukemias exhibit an abnormal increase of monocytic cells. These effectscould possibly be prevented by antagonists to zcytor17, such asdescribed herein. Moreover, anti-zcytor17 antibodies and bindingpartners can be conjugated to molecules such as toxic moieties andcytokines, as described herein to direct the killing of leukemiamonocytic cells.

[0127] Using methods known in the art, and disclosed herein, one ofskill could readily assess the activity of zcytor17 agonists andantagonists in the disease states disclosed herein, inflammation,cancer, or infection as well as other disease states involving monocyticcells. In addition, as zcytor17 is expressed in a monocyte-specificmanner, and these diseases involve abnormalities in monocytic cells,such as cell proliferation, function, localization, and activation, thepolynucleotides, polypeptides, and antibodies of the present inventioncan be used to as diagnostics to detect such monocytic cellabnormalities, and indicate the presence of disease. Such methodsinvolve taking a biological sample from a patient, such as blood,saliva, or biopsy, and comparing it to a normal control sample.Histological, cytological, flow cytometric, biochemical and othermethods can be used to determine the relative levels or localization ofzcytor17, or cells expressing zcytor17, i.e., monocytes, in the patientsample compared to the normal control. A change in the level (increaseor decrease) of zcytor17 expression, or a change in number orlocalization of monocytes (e.g., increase or infiltration of monocyticcells in tissues where they are not normally present) compared to acontrol would be indicative of disease. Such diagnostic methods can alsoinclude using radiometric, fluorescent, and colorimetric tags attachedto polynucleotides, polypeptides or antibodies of the present invention.Such methods are well known in the art and disclosed herein.

[0128] Amino acid sequences having Zcytor17 activity can be used tomodulate the immune system by binding Zcytor17 ligand, and thus,preventing the binding of Zcytor17 ligand with endogenous Zcytor17receptor. Zcytor17 antagonists, such as anti-Zcytor17 antibodies, canalso be used to modulate the immune system by inhibiting the binding ofZcytor17 ligand with the endogenous Zcytor17 receptor. Accordingly, thepresent invention includes the use of proteins, polypeptides, andpeptides having Zcytor17 activity (such as Zcytor17 polypeptides,Zcytor17 analogs (e.g., anti-Zcytor17 anti-idiotype antibodies), andZcytor17 fusion proteins) to a subject which lacks an adequate amount ofthis polypeptide, or which produces an excess of Zcytor17 ligand.Zcytor17 antagonists (e.g., anti-Zcytor17 antibodies) can be also usedto treat a subject which produces an excess of either Zcytor17 ligand orZcytor17. Suitable subjects include mammals, such as humans.

[0129] Zcytor17 has been shown to be upregulated in monocyte cells, andmay be involved in regulating inflammation. As such, polypeptides of thepresent invention can be assayed and used for their ability to modifyinflammation, or can be used as a marker for inflammation. Methods todetermine proinflammatory and antiinflammatory qualities of zcytor17 areknown in the art and discussed herein. Moreover, it may be involved inup-regulating the production of acute phase reactants, such as serumamyloid A (SAA), α1-antichymotrypsin, and haptoglobin, and thatexpression of zcytor17 ligand may be increased upon injection oflipopolysaccharide (LPS) in vivo that are involved in inflammatoryresponse (Dumoutier, L. et al., Proc. Nat'l. Acad. Sci. 97:10144-10149,2000). Production of acute phase proteins, such as SAA, is considered sshort-term survival mechanism where inflammation is beneficial; however,maintenance of acute phase proteins for longer periods contributes tochronic inflammation and can be harmful to human health. For review, seeUhlar, C M and Whitehead, A S, Eur. J. Biochem. 265:501-523, 1999, andBaumann H. and Gauldie, J. Immunology Today 15:74-80, 1994. Moreover,the acute phase protein SAA is implicated in the pathogenesis of severalchronic inflammatory diseases, is implicated in atherosclerosis andrheumatoid arthritis, and is the precursor to the amyloid A proteindeposited in amyloidosis (Uhlar, C M and Whitehead, supra.). Thus, wherea ligand for zcytor17 that acts as a pro-inflammatory molecule andinduces production of SAA, antagonists would be useful in treatinginflammatory disease and other diseases associated with acute phaseresponse proteins induced by the ligand. Such antagonists are providedby the present invention. For example, a method of reducing inflammationcomprises administering to a mammal with inflammation an amount of acomposition of soluble zcytor17-comprising receptor, or anti-zcytor17antibody that is sufficient to reduce inflammation. Moreover, a methodof suppressing an inflammatory response in a mammal with inflammationcan comprise: (1) determining a level of serum amyloid A protein; (2)administering a composition comprising a soluble zcytor17 cytokinereceptor polypeptide as described herein in an acceptable pharmaceuticalvehicle; (3) determining a post administration level of serum amyloid Aprotein; (4) comparing the level of serum amyloid A protein in step (1)to the level of serum amyloid A protein in step (3), wherein a lack ofincrease or a decrease in serum amyloid A protein level is indicative ofsuppressing an inflammatory response.

[0130] The receptors of the present invention include at least onezcytor17 receptor subunit. A second receptor polypeptide included in theheterodimeric soluble receptor belongs to the receptor subfamily thatincludes class I cytokine receptor subunits. According to the presentinvention, in addition to a monomeric or homodimeric zcytor17 receptorpolypeptide, a heterodimeric soluble zcytor17 receptor, as exemplifiedby an embodiment comprising a soluble zcytor17 receptor+soluble Class Ireceptor heterodimeric component, can act as an antagonist of thenatural zcytor17 ligand. Other embodiments include soluble multimericreceptors comprising zcytor17.

[0131] Analysis of the tissue distribution of the mRNA correspondingzcytor17 cDNA showed that mRNA level was highest in monocytes andprostate cells, and is elevated in activated monocytes, and activatedCD4+, activated CD8+, and activated CD3+ cells. Hence, zcytor17 isimplicated in inducing inflammatory and immune response. Thus,particular embodiments of the present invention are directed toward useof soluble zcytor17 heterodimers as antagonists in inflammatory andimmune diseases or conditions such as pancreatitis, type I diabetes(IDDM), pancreatic cancer, pancreatitis, Graves Disease, inflammatorybowel disease (IBD), Crohn's Disease, colon and intestinal cancer,diverticulosis, autoimmune disease, sepsis, organ or bone marrowtransplant; inflammation due to trauma, sugery or infection;amyloidosis; splenomegaly; graft versus host disease; and whereinhibition of inflammation, immune suppression, reduction ofproliferation of hematopoietic, immune, inflammatory or lymphoid cells,macrophages, T-cells (including Th1 and Th2 cells, CD4+ and CD8+ cells),suppression of immune response to a pathogen or antigen. Moreover thepresence of zcytor178 expression in activated immune cells such asactivated CD4+ and CD19+ cells showed that zcytor17 may be involved inthe body's immune defensive reactions against foreign invaders: such asmicroorganisms and cell debris, and could play a role in immuneresponses during inflammation and cancer formation. As such, antibodiesand bidning partners of the present invention that are agonistic orantagonistic to zcytor17 function, can be used to modify immune responseand inflammation.

[0132] Moreover, antibodies or binding polypeptides that bind zcytor17polypeptides, monomers, homodimers, heterodimers and multimers describedherein and/or zcytor17 polypeptides, monomers, homodimers, heterodimersand multimers themselves are useful to:

[0133] 1) Antagonize or block signaling via the zcytor17 receptors inthe treatment of acute inflammation, inflammation as a result of trauma,tissue injury, surgery, sepsis or infection, and chronic inflammatorydiseases such as asthma, inflammatory bowel disease (IBD), chroniccolitis, splenomegaly, rheumatoid arthritis, recurrent acuteinflammatory episodes (e.g., tuberculosis), and treatment ofamyloidosis, and atherosclerosis, Castleman's Disease, asthma, and otherdiseases associated with the induction of acute-phase response.

[0134] 2) Antagonize or block signaling via the zcytor17 receptors inthe treatment of autoimmune diseases such as IDDM, multiple sclerosis(MS), systemic Lupus erythematosus (SLE), myasthenia gravis, rheumatoidarthritis, and IBD to prevent or inhibit signaling in immune cells (e.g.lymphocytes, monocytes, leukocytes) via zcytor17 (Hughes C et al., J.Immunol 153: 3319-3325, 1994). Alternatively antibodies, such asmonoclonal antibodies (MAb) to zcytor17-comprising receptors, can alsobe used as an antagonist to deplete unwanted immune cells to treatautoimmune disease. Asthma, allergy and other atopic disease may betreated with an MAb against, for example, soluble zcytor17 solublereceptors or zcytor17/CRF2-4 heterodimers, to inhibit the immuneresponse or to deplete offending cells. Blocking or inhibiting signalingvia zcytor17, using the polypeptides and antibodies of the presentinvention, may also benefit diseases of the pancreas, kidney, pituitaryand neuronal cells. IDDM, NIDDM, pancreatitis, and pancreatic carcinomamay benefit. Zcytor17 may serve as a target for MAb therapy of cancerwhere an antagonizing MAb inhibits cancer growth and targetsimmune-mediated killing. (Holliger P, and Hoogenboom, H: Nature Biotech.16: 1015-1016, 1998). Mabs to soluble zcytor17 monomers, homodimers,heterodimers and multimers may also be useful to treat nephropathiessuch as glomerulosclerosis, membranous neuropathy, amyloidosis (whichalso affects the kidney among other tissues), renal arteriosclerosis,glomerulonephritis of various origins, fibroproliferative diseases ofthe kidney, as well as kidney dysfunction associated with SLE, IDDM,type II diabetes (NIDDM), renal tumors and other diseases.

[0135] 3) Agonize or initiate signaling via the zcytor17 receptors inthe treatment of autoimmune diseases such as IDDM, MS, SLE, myastheniagravis, rheumatoid arthritis, and IBD. Anti-zcytor17, anti-heterodimerand multimer monoclonal antibodies may signal lymphocytes or otherimmune cells to differentiate, alter proliferation, or change productionof cytokines or cell surface proteins that ameliorate autoimmunity.Specifically, modulation of a T-helper cell response to an alternatepattern of cytokine secretion may deviate an autoimmune response toameliorate disease (Smith J A et al., J. Immunol. 160:4841-4849, 1998).Similarly, agonistic anti-zcytor17, anti-heterodimer and multimermonoclonal antibodies may be used to signal, deplete and deviate immunecells involved in asthma, allergy and atopoic disease. Signaling viazcytor17 may also benefit diseases of the pancreas, kidney, pituitaryand neuronal cells. IDDM, NIDDM, pancreatitis, and pancreatic carcinomamay benefit. Zcytor17 may serve as a target for MAb therapy ofpancreatic cancer where a signaling MAb inhibits cancer growth andtargets immune-mediated killing (Tutt, A L et al., J Immunol. 161:3175-3185, 1998). Similarly T-cell specific leukemias, lymphomas, andcarcinoma may be treated with monoclonal antibodies tozcytor17-comprising soluble receptors of the present invention.

[0136] Soluble zcytor17 monomeric, homodimeric, heterodimeric andmultimeric polypeptides described herein can be used to neutralize/blockzcytor17 ligand activity in the treatment of autoimmune disease, atopicdisease, NIDDM, pancreatitis and kidney dysfunction as described above.A soluble form of zcytor17 may be used to promote an antibody responsemediated by T cells and/or to promote the production of IL-4 or othercytokines by lymphocytes or other immune cells.

[0137] The soluble zcytor17-comprising receptors of the presentinvention are useful as antagonists of its natural ligand. Suchantagonistic effects can be achieved by direct neutralization or bindingof its natural ligand. In addition to antagonistic uses, the solublereceptors of the present invention can bind zcytor17 ligand and act ascarrier proteins for the ligand, in order to transport the ligand todifferent tissues, organs, and cells within the body. As such, thesoluble receptors of the present invention can be fused or coupled tomolecules, polypeptides or chemical moieties that direct thesoluble-receptor-Ligand complex to a specific site, such as a tissue,specific immune cell, monocytes, or tumor. For example, in acuteinfection or some cancers, benefit may result from induction ofinflammation and local acute phase response proteins. Thus, the solublereceptors of the present invention can be used to specifically directthe action of a pro-inflammatory ligand. See, Cosman, D. Cytokine 5:95-106, 1993; and Fernandez-Botran, R. Exp. Opin. Invest. Drugs9:497-513, 2000.

[0138] Moreover, the soluble receptors of the present invention can beused to stabilize the zcytor17 ligand, to increase the bioavailability,therapeutic longevity, and/or efficacy of the Ligand by stabilizing theLigand from degradation or clearance, or by targeting the ligand to asite of action within the body. For example the naturally occurringIL-6/soluble IL-6R complex stabilizes IL-6 and can signal through thegp130 receptor. See, Cosman, D. supra., and Fernandez-Botran, R. supra.Moreover, Zcytor17 may be combined with a cognate ligand such as itsligand to comprise a ligand/soluble receptor complex. Such complexes maybe used to stimulate responses from cells presenting a companionreceptor subunit. The cell specificity of zcytor17/ligand complexes maydiffer from that seen for the ligand administered alone. Furthermore thecomplexes may have distinct pharmacokinetic properties such as affectinghalf-life, dose/response and organ or tissue specificity.Zcytor17/ligand complexes thus may have agonist activity to enhance animmune response or stimulate mesangial cells or to stimulate hepaticcells. Alternatively only tissues expressing a signaling subunit theheterodimerizes with the complex may be affected analogous to theresponse to IL6/IL6R complexes (Hirota H. et al., Proc. Nat'l. Acad.Sci. 92:4862-4866, 1995; Hirano, T. in Thomason, A. (Ed.) “The CytokineHandbook”, 3^(rd) Ed., p. 208-209). Soluble receptor/cytokine complexesfor IL12 and CNTF display similar activities.

[0139] Zcytor17 homodimeric, heterodimeric and multimeric receptorpolypeptides may also be used within diagnostic systems for thedetection of circulating levels of ligand, and in the detection of acutephase inflammatory response. Within a related embodiment, antibodies orother agents that specifically bind to Zcytor17 soluble receptors of thepresent invention can be used to detect circulating receptorpolypeptides; conversely, Zcytor17 soluble receptors themselves can beused to detect circulating or locally-acting ligand polypeptides.Elevated or depressed levels of ligand or receptor polypeptides may beindicative of pathological conditions, including inflammation or cancer.Moreover, detection of acute phase proteins or molecules such aszcytor17 ligand can be indicative of a chronic inflammatory condition incertain disease states (e.g., rheumatoid arthritis). Detection of suchconditions serves to aid in disease diagnosis as well as help aphysician in choosing proper therapy.

[0140] Differentiation is a progressive and dynamic process, beginningwith pluripotent stem cells and ending with terminally differentiatedcells. Pluripotent stem cells that can regenerate without commitment toa lineage express a set of differentiation markers that are lost whencommitment to a cell lineage is made. Progenitor cells express a set ofdifferentiation markers that may or may not continue to be expressed asthe cells progress down the cell lineage pathway toward maturation.Differentiation markers that are expressed exclusively by mature cellsare usually functional properties such as cell products, enzymes toproduce cell products, and receptors. The stage of a cell population'sdifferentiation is monitored by identification of markers present in thecell population. Myocytes, osteoblasts, adipocytes, chrondrocytes,fibroblasts and reticular cells are believed to originate from a commonmesenchymal stem cell (Owen et al., Ciba Fdn. Symp. 136:42-46, 1988).Markers for mesenchymal stem cells have not been well identified (Owenet al., J. of Cell Sci. 87:731-738, 1987), so identification is usuallymade at the progenitor and mature cell stages. The novel polypeptides ofthe present invention may be useful for studies to isolate mesenchymalstem cells and myocyte or other progenitor cells, both in vivo and exvivo.

[0141] There is evidence to suggest that factors that stimulate orregulate specific cell types down a pathway towards terminaldifferentiation or dedifferentiation affect the entire cell populationoriginating from a common precursor or stem cell. Thus, the presentinvention includes stimulating or inhibiting the proliferation oflymphoid cells, hematopoietic cells and endothelial cells. Thusmolecules of the present invention, such as soluble zcytor17 receptors,cytokine-binding fragments, anti-zcytor17 antibodies, sense andantisense polynucleotides may have use in inhibiting tumor cells, andparticularly lymphoid, hematopoietic, prostate, endothelial, and thyroidtumor cells.

[0142] Assays measuring differentiation include, for example, measuringcell markers associated with stage-specific expression of a tissue,enzymatic activity, functional activity or morphological changes (Watt,FASEB; 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes,Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; allincorporated herein by reference). Alternatively, zcytor17 polypeptideitself can serve as an additional cell-surface or secreted markerassociated with stage-specific expression of a tissue. As such, directmeasurement of zcytor17 polypeptide, or its loss of expression in atissue as it differentiates, can serve as a marker for identification ordifferentiation of, e.g., prostate tissue, or monocyte cells.

[0143] Similarly, direct measurement of zcytor17 polypeptide, or itsloss of expression in a tissue can be determined in a tissue or cells asthey undergo tumor progression. Increases in invasiveness and motilityof cells, or the gain or loss of expression of zcytor17 in apre-cancerous or cancerous condition, in comparison to normal tissue,can serve as a diagnostic for transformation, invasion and metastasis intumor progression. As such, knowledge of a tumor's stage of progressionor metastasis will aid the physician in choosing the most propertherapy, or aggressiveness of treatment, for a given individual cancerpatient. Methods of measuring gain and loss of expression (of eithermRNA or protein) are well known in the art and described herein and canbe applied to zcytor17 expression. For example, appearance ordisappearance of polypeptides that regulate cell motility can be used toaid diagnosis and prognosis of prostate cancer (Banyard, J. and Zetter,B. R., Cancer and Metast. Rev. 17:449-458, 1999). As an effector of cellmotility, activation, proliferation, or differentiation, zcytor17 gainor loss of expression may serve as a diagnostic for lymphoid,hematopoietic, prostate, endothelial, and thyroid and other cancers.

[0144] In addition, as zcytor17 is monocyte and prostate-specific,polynucleotide probes, anti-zcytor17 antibodies, and detection thepresence of zcytor17 polypeptides in tissues can be used to assesswhether monocytes or prostate tissue is present, for example, aftersurgery involving the excision of a diseased or cancerous prostate, orin evaluation of monocyte infiltration in diseased or infected tissuesor monocyte cancers. As such, the polynucleotides, polypeptides, andantibodies of the present invention can be used as an aid to determinewhether all prostate tissue is excised after surgery, for example, aftersurgery for prostate cancer. In such instances, it is especiallyimportant to remove all potentially diseased tissue to maximize recoveryfrom the cancer, and to minimize recurrence. Moreover, thepolynucleotides, polypeptides, and antibodies of the present inventioncan be used as an aid to determine whether monocyte infiltration ispresent in diseased tissues (e.g., inflamed or infected) to monitor therecovery from disease or cancers. Preferred embodiments includefluorescent, radiolabeled, or calorimetrically labeled anti-zcytor17antibodies and zcytor17 polypeptide binding partners, that can be usedhistologically or in situ.

[0145] Moreover, the activity and effect of zcytor17 on tumorprogression and metastasis can be measured in vivo. Several syngeneicmouse models have been developed to study the influence of polypeptides,compounds or other treatments on tumor progression. In these models,tumor cells passaged in culture are implanted into mice of the samestrain as the tumor donor. The cells will develop into tumors havingsimilar characteristics in the recipient mice, and metastasis will alsooccur in some of the models. Appropriate tumor models for our studiesinclude the Lewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma(ATCC No. CRL-6323), amongst others. These are both commonly used tumorlines, syngeneic to the C57BL6 mouse, that are readily cultured andmanipulated in vitro. Tumors resulting from implantation of either ofthese cell lines are capable of metastasis to the lung in C57BL6 mice.The Lewis lung carcinoma model has recently been used in mice toidentify an inhibitor of angiogenesis (O'Reilly M S, et al. Cell 79:315-328,1994). C57BL6/J mice are treated with an experimental agenteither through daily injection of recombinant protein, agonist orantagonist or a one time injection of recombinant adenovirus. Three daysfollowing this treatment, 10⁵ to 10⁶ cells are implanted under thedorsal skin. Alternatively, the cells themselves may be infected withrecombinant adenovirus, such as one expressing zcytor17, beforeimplantation so that the protein is synthesized at the tumor site orintracellularly, rather than systemically. The mice normally developvisible tumors within 5 days. The tumors are allowed to grow for aperiod of up to 3 weeks, during which time they may reach a size of1500-1800 mm³ in the control treated group. Tumor size and body weightare carefully monitored throughout the experiment. At the time ofsacrifice, the tumor is removed and weighed along with the lungs and theliver. The lung weight has been shown to correlate well with metastatictumor burden. As an additional measure, lung surface metastases arecounted. The resected tumor, lungs and liver are prepared forhistopathological examination, immunohistochemistry, and in situhybridization, using methods known in the art and described herein. Theinfluence of the expressed polypeptide in question, e.g., zcytor17, onthe ability of the tumor to recruit vasculature and undergo metastasiscan thus be assessed. In addition, aside from using adenovirus, theimplanted cells can be transiently transfected with zcytor17. Use ofstable zcytor17 transfectants as well as use of induceable promoters toactivate zcytor17 expression in vivo are known in the art and can beused in this system to assess zcytor17 induction of metastasis.Moreover, purified zcytor17 or zcytor17 conditioned media can bedirectly injected in to this mouse model, and hence be used in thissystem. For general reference see, O'Reilly M S, et al. Cell 79:315-328,1994; and Rusciano D, et al. Murine Models of Liver Metastasis. InvasionMetastasis 14:349-361, 1995.

[0146] The activity of zcytor17 and its derivatives (conjugates) ongrowth and dissemination of tumor cells derived from human hematologicmalignancies can also be measured in vivo in a mouse Xenograft modelSeveral mouse models have been developed in which human tumor cells areimplanted into immunodeficient mice, collectively referred to asxenograft models. See Cattan, A R and Douglas, E Leuk. Res. 18:513-22,1994; and Flavell, D J, Hematological Oncology 14:67-82, 1996. Thecharacteristics of the disease model vary with the type and quantity ofcells delivered to the mouse. Typically, the tumor cells willproliferate rapidly and can be found circulating in the blood andpopulating numerous organ systems. Therapeutic strategies appropriatefor testing in such a model include antibody induced toxicity,ligand-toxin conjugates or cell-based therapies. The latter method,commonly referred to adoptive immunotherapy, involves treatment of theanimal with components of the human immune system (i.e. lymphocytes, NKcells) and may include ex vivo incubation of cells with zcytor17 orother immunomodulatory agents.

[0147] The mRNA corresponding to this novel DNA showed expression inlymphoid tissues, including thymus, is expressed in bone marrow andprostate, monocytes, and activated monocytes, CD19+ B-cells, and may beexpressed in spleen, lymph nodes, and peripheral blood leukocytes. Thesedata indicate a role for the zcytor17 receptor in proliferation,differentiation, and/or activation of immune cells, and suggest a rolein development and regulation of immune responses. The data also suggestthat the interaction of zcytor17 with its ligand may stimulateproliferation and development of myeloid cells and may, like IL-2, IL-6,LIF, IL-11, IL-12 and OSM (Baumann et al., J. Biol. Chem. 268:8414-8417,1993), induce acute-phase protein synthesis in hepatocytes.

[0148] It is preferred to purify the polypeptides of the presentinvention to ≧80% purity, more preferably to ≧90% purity, even morepreferably ≧95% purity, and particularly preferred is a pharmaceuticallypure state, that is greater than 99.9% pure with respect tocontaminating macromolecules, particularly other proteins and nucleicacids, and free of infectious and pyrogenic agents. Preferably, apurified polypeptide is substantially free of other polypeptides,particularly other polypeptides of animal origin.

[0149] Expressed recombinant zcytor17 polypeptides (or zcytor17 chimericor fusion polypeptides) can be purified using fractionation and/orconventional purification methods and media. Ammonium sulfateprecipitation and acid or chaotrope extraction may be used forfractionation of samples. Exemplary purification steps may includehydroxyapatite, size exclusion, FPLC and reverse-phase high performanceliquid chromatography. Suitable chromatographic media includederivatized dextrans, agarose, cellulose, polyacrylamide, specialtysilicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.Exemplary chromatographic media include those media derivatized withphenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding receptor polypeptides tosupport media are well known in the art. Selection of a particularmethod is a matter of routine design and is determined in part by theproperties of the chosen support. See, for example, AffinityChromatography: Principles & Methods, Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988.

[0150] The polypeptides of the present invention can be isolated byexploitation of their biochemical, structural, and biologicalproperties. For example, immobilized metal ion adsorption (IMAC)chromatography can be used to purify histidine-rich proteins, includingthose comprising polyhistidine tags. Briefly, a gel is first chargedwith divalent metal ions to form a chelate (Sulkowski, Trends inBiochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to thismatrix with differing affinities, depending upon the metal ion used, andwill be eluted by competitive elution, lowering the pH, or use of strongchelating agents. Other methods of purification include purification ofglycosylated proteins by lectin affinity chromatography and ion exchangechromatography (Methods in Enzymol., Vol. 182, “Guide to ProteinPurification”, M. Deutscher, (ed.), Acad. Press, San Diego, 1990,pp.529-39). Within additional embodiments of the invention, a fusion ofthe polypeptide of interest and an affinity tag (e.g., maltose-bindingprotein, an immunoglobulin domain) may be constructed to facilitatepurification.

[0151] Moreover, using methods described in the art, polypeptidefusions, or hybrid zcytor17 proteins, are constructed using regions ordomains of the inventive zcytor17 in combination with those of otherhuman cytokine receptor family proteins, or heterologous proteins(Sambrook et al., ibid., Altschul et al., ibid., Picard, Cur. Opin.Biology, 5:511-5, 1994, and references therein). These methods allow thedetermination of the biological importance of larger domains or regionsin a polypeptide of interest. Such hybrids may alter reaction kinetics,binding, constrict or expand the substrate specificity, or alter tissueand cellular localization of a polypeptide, and can be applied topolypeptides of unknown structure.

[0152] Fusion polypeptides or proteins can be prepared by methods knownto those skilled in the art by preparing each component of the fusionprotein and chemically conjugating them. Alternatively, a polynucleotideencoding one or more components of the fusion protein in the properreading frame can be generated using known techniques and expressed bythe methods described herein. For example, part or all of a domain(s)conferring a biological function may be swapped between zcytor17 of thepresent invention with the functionally equivalent domain(s) fromanother cytokine family member. Such domains include, but are notlimited to, the secretory signal sequence, extracellular domain,cytokine binding domain, fibronectin type III domain, transmembranedomain, and intracellular signaling domain, Box I and Box II sites, asdisclosed herein. Such fusion proteins would be expected to have abiological functional profile that is the same or similar topolypeptides of the present invention or other known family proteins,depending on the fusion constructed. Moreover, such fusion proteins mayexhibit other properties as disclosed herein.

[0153] Standard molecular biological and cloning techniques can be usedto swap the equivalent domains between the zcytor17 polypeptide andthose polypeptides to which they are fused. Generally, a DNA segmentthat encodes a domain of interest, e.g., a zcytor17 domain describedherein, is operably linked in frame to at least one other DNA segmentencoding an additional polypeptide (for instance a domain or region fromanother cytokine receptor, such as the gp130, LIF, IL-12, WSX-1, IL-2 orother class I cytokine receptor), and inserted into an appropriateexpression vector, as described herein. Generally DNA constructs aremade such that the several DNA segments that encode the correspondingregions of a polypeptide are operably linked in frame to make a singleconstruct that encodes the entire fusion protein, or a functionalportion thereof. For example, a DNA construct would encode fromN-terminus to C-terminus a fusion protein comprising a signalpolypeptide followed by a cytokine binding domain, followed by atransmembrane domain, followed by an intracellular signaling domain.Such fusion proteins can be expressed, isolated, and assayed foractivity as described herein. Moreover, such fusion proteins can be usedto express and secrete fragments of the zcytor17 polypeptide, to beused, for example to inoculate an animal to generate anti-zcytor17antibodies as described herein. For example a secretory signal sequencecan be operably linked to the cytokine binding domain, transmembranedomain, intracellular signaling domain or subfragment thereof, or acombination thereof (e.g., operably linked polypeptides comprising theextracellular cytokine binding domain fused to a transmembrane domain,or zcytor17 polypeptide fragments described herein), to secrete afragment of zcytor17 polypeptide that can be purified as describedherein and serve as an antigen to be inoculated into an animal toproduce anti-zytor 17 antibodies, as described herein.

[0154] Zcytor17 polypeptides or fragments thereof may also be preparedthrough chemical synthesis. zcytor17 polypeptides may be monomers ormultimers; glycosylated or non-glycosylated; pegylated or non-pegylated;and may or may not include an initial methionine amino acid residue.

[0155] Polypeptides of the present invention can also be synthesized byexclusive solid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. Methods for synthesizingpolypeptides are well known in the art. See, for example, Merrifield, J.Am. Chem. Soc. 85:2149, 1963; Kaiser et al., Anal. Biochem. 34:595,1970. After the entire synthesis of the desired peptide on a solidsupport, the peptide-resin is with a reagent which cleaves thepolypeptide from the resin and removes most of the side-chain protectinggroups. Such methods are well established in the art.

[0156] The activity of molecules of the present invention can bemeasured using a variety of assays that measure cell differentiation andproliferation. Such assays are well known in the art.

[0157] Proteins of the present invention are useful for example, intreating and diagnosing lymphoid, immune, inflammatory, spleenic, bloodor bone disorders, and can be measured in vitro using cultured cells orin vivo by administering molecules of the present invention to theappropriate animal model. For instance, host cells expressing a zcytor17soluble receptor polypeptide can be embedded in an alginate environmentand injected (implanted) into recipient animals. Alginate-poly-L-lysinemicroencapsulation, permselective membrane encapsulation and diffusionchambers are a means to entrap transfected mammalian cells or primarymammalian cells. These types of non-immunogenic “encapsulations” permitthe diffusion of proteins and other macromolecules secreted or releasedby the captured cells to the recipient animal. Most importantly, thecapsules mask and shield the foreign, embedded cells from the recipientanimal's immune response. Such encapsulations can extend the life of theinjected cells from a few hours or days (naked cells) to several weeks(embedded cells). Alginate threads provide a simple and quick means forgenerating embedded cells.

[0158] The materials needed to generate the alginate threads are knownin the art. In an exemplary procedure, 3% alginate is prepared insterile H₂O, and sterile filtered. Just prior to preparation of alginatethreads, the alginate solution is again filtered. An approximately 50%cell suspension (containing about 5×10⁵ to about 5×10⁷ cells/ml) ismixed with the 3% alginate solution. One ml of the alginate/cellsuspension is extruded into a 100 mM sterile filtered CaCl₂ solutionover a time period of ˜15 min, forming a “thread”. The extruded threadis then transferred into a solution of 50 mM CaCl₂, and then into asolution of 25 mM CaCl₂. The thread is then rinsed with deionized waterbefore coating the thread by incubating in a 0.01% solution ofpoly-L-lysine. Finally, the thread is rinsed with Lactated Ringer'sSolution and drawn from solution into a syringe barrel (without needle).A large bore needle is then attached to the syringe, and the thread isintraperitoneally injected into a recipient in a minimal volume of theLactated Ringer's Solution.

[0159] An in vivo approach for assaying proteins of the presentinvention involves viral delivery systems. Exemplary viruses for thispurpose include adenovirus, herpesvirus, retroviruses, vaccinia virus,and adeno-associated virus (AAV). Adenovirus, a double-stranded DNAvirus, is currently the best studied gene transfer vector for deliveryof heterologous nucleic acid (for review, see T. C. Becker et al., Meth.Cell Biol. 43:161-89, 1994; and J. T. Douglas and D. T. Curiel, Science& Medicine 4:44-53, 1997). The adenovirus system offers severaladvantages: (i) adenovirus can accommodate relatively large DNA inserts;(ii) can be grown to high-titer; (iii) infect a broad range of mammaliancell types; and (iv) can be used with a large number of differentpromoters including ubiquitous, tissue specific, and regulatablepromoters. Also, because adenoviruses are stable in the bloodstream,they can be administered by intravenous injection.

[0160] Using adenovirus vectors where portions of the adenovirus genomeare deleted, inserts are incorporated into the viral DNA by directligation or by homologous recombination with a co-transfected plasmid.In an exemplary system, the essential E1 gene has been deleted from theviral vector, and the virus will not replicate unless the E1 gene isprovided by the host cell (the human 293 cell line is exemplary). Whenintravenously administered to intact animals, adenovirus primarilytargets the liver. If the adenoviral delivery system has an E1 genedeletion, the virus cannot replicate in the host cells. However, thehost's tissue (e.g., liver) will express and process (and, if asecretory signal sequence is present, secrete) the heterologous protein.Secreted proteins will enter the circulation in the highly vascularizedliver, and effects on the infected animal can be determined.

[0161] Moreover, adenoviral vectors containing various deletions ofviral genes can be used in an attempt to reduce or eliminate immuneresponses to the vector. Such adenoviruses are E1 deleted, and inaddition contain deletions of E2A or E4 (Lusky, M. et al., J. Virol.72:2022-2032, 1998; Raper, S. E. et al., Human Gene Therapy 9:671-679,1998). In addition, deletion of E2b is reported to reduce immuneresponses (Amalfitano, A. et al., J. Virol. 72:926-933, 1998). Moreover,by deleting the entire adenovirus genome, very large inserts ofheterologous DNA can be accommodated. Generation of so called “gutless”adenoviruses where all viral genes are deleted are particularlyadvantageous for insertion of large inserts of heterologous DNA. Forreview, see Yeh, P. and Perricaudet, M., FASEB J. 11:615-623, 1997.

[0162] The adenovirus system can also be used for protein production invitro. By culturing adenovirus-infected non-293 cells under conditionswhere the cells are not rapidly dividing, the cells can produce proteinsfor extended periods of time. For instance, BHK cells are grown toconfluence in cell factories, then exposed to the adenoviral vectorencoding the secreted protein of interest. The cells are then grownunder serum-free conditions, which allows infected cells to survive forseveral weeks without significant cell division. Alternatively,adenovirus vector infected 293 cells can be grown as adherent cells orin suspension culture at relatively high cell density to producesignificant amounts of protein (See Garnier et al., Cytotechnol.15:145-55, 1994). With either protocol, an expressed, secretedheterologous protein can be repeatedly isolated from the cell culturesupernatant, lysate, or membrane fractions depending on the dispositionof the expressed protein in the cell. Within the infected 293 cellproduction protocol, non-secreted proteins may also be effectivelyobtained.

[0163] In view of the tissue distribution observed for zcytor17,agonists (including the natural ligand/substrate/cofactor/etc.) andantagonists have enormous potential in both in vitro and in vivoapplications. Compounds identified as zcytor17 agonists are useful forstimulating growth of immune and hematopoietic cells in vitro and invivo. For example, zcytor17 soluble receptors, and agonist compounds areuseful as components of defined cell culture media, and may be usedalone or in combination with other cytokines and hormones to replaceserum that is commonly used in cell culture. Agonists are thus useful inspecifically promoting the growth and/or development of T-cells,B-cells, and other cells of the lymphoid and myeloid lineages inculture. Moreover, zcytor17 soluble receptor, agonist, or antagonist maybe used in vitro in an assay to measure stimulation of colony formationfrom isolated primary bone marrow cultures. Such assays are well knownin the art.

[0164] Antagonists are also useful as research reagents forcharacterizing sites of ligand-receptor interaction. Inhibitors ofzcytor17 activity (zcytor17 antagonists) include anti-zcytor17antibodies and soluble zcytor17 receptors, as well as other peptidic andnon-peptidic agents (including ribozymes).

[0165] Zcytor17 can also be used to identify modulators (e.g,antagonists) of its activity. Test compounds are added to the assaysdisclosed herein to identify compounds that inhibit the activity ofzcytor17. In addition to those assays disclosed herein, samples can betested for inhibition of zcytor17 activity within a variety of assaysdesigned to measure zcytor17 binding, oligomerization, or thestimulation/inhibition of zcytor17-dependent cellular responses. Forexample, zcytor17-expressing cell lines can be transfected with areporter gene construct that is responsive to a zcytor17-stimulatedcellular pathway. Reporter gene constructs of this type are known in theart, and will generally comprise a zcytor17-DNA response elementoperably linked to a gene encoding an assay detectable protein, such asluciferase. DNA response elements can include, but are not limited to,cyclic AMP response elements (CRE), hormone response elements (HRE)insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci.USA 87:5273-7, 1990) and serum response elements (SRE) (Shaw et al. Cell56: 563-72, 1989). Cyclic AMP response elements are reviewed in Roestleret al., J. Biol. Chem. 263 (19):9063-6; 1988 and Habener, Molec.Endocrinol. 4 (8):1087-94; 1990. Hormone response elements are reviewedin Beato, Cell 56:335-44; 1989. Candidate compounds, solutions, mixturesor extracts or conditioned media from various cell types are tested forthe ability to enhance the activity of zcytor17 receptor as evidenced bya increase in zcytor17 stimulation of reporter gene expression. Assaysof this type will detect compounds that directly stimulate zcytor17signal transduction activity through binding the receptor or byotherwise stimulating part of the signal cascade. As such, there isprovided a method of identifying agonists of zcytor17 polypeptide,comprising providing cells responsive to a zcytor17 polypeptide,culturing a first portion of the cells in the absence of a testcompound, culturing a second portion of the cells in the presence of atest compound, and detecting a increase in a cellular response of thesecond portion of the cells as compared to the first portion of thecells. Moreover third cell, containing the reporter gene constructdescribed above, but not expressing zcytor17 receptor, can be used as acontrol cell to assess non-specific, or non-zcytor17-mediated,stimulation of the reporter. Agonists, including the natural ligand, aretherefore useful to stimulate or increase zcytor17 polypeptide function.

[0166] A zcytor17 ligand-binding polypeptide, such as the extracellulardomain or cytokine binding domain disclosed herein, can also be used forpurification of ligand. The polypeptide is immobilized on a solidsupport, such as beads of agarose, cross-linked agarose, glass,cellulosic resins, silica-based resins, polystyrene, cross-linkedpolyacrylamide, or like materials that are stable under the conditionsof use. Methods for linking polypeptides to solid supports are known inthe art, and include amine chemistry, cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, and hydrazide activation. The resulting medium willgenerally be configured in the form of a column, and fluids containingligand are passed through the column one or more times to allow ligandto bind to the receptor polypeptide. The ligand is then eluted usingchanges in salt concentration, chaotropic agents (guanidine HCl), or pHto disrupt ligand-receptor binding.

[0167] An assay system that uses a ligand-binding receptor (or anantibody, one member of a complement/anti-complement pair) or a bindingfragment thereof, and a commercially available biosensor instrument maybe advantageously employed (e.g., BIAcore™, Pharmacia Biosensor,Piscataway, N.J.; or SELD™ technology, Ciphergen, Inc., Palo Alto,Calif.). Such receptor, antibody, member of a complement/anti-complementpair or fragment is immobilized onto the surface of a receptor chip. Useof this instrument is disclosed by Karlsson, J. Immunol. Methods145:229-240, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63,1993. A receptor, antibody, member or fragment is covalently attached,using amine or sulfhydryl chemistry, to dextran fibers that are attachedto gold film within the flow cell. A test sample is passed through thecell. If a ligand, epitope, or opposite member of thecomplement/anti-complement pair is present in the sample, it will bindto the immobilized receptor, antibody or member, respectively, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination of on- and off-rates, from which binding affinity canbe calculated, and assessment of stoichiometry of binding.

[0168] Ligand-binding receptor polypeptides can also be used withinother assay systems known in the art. Such systems include Scatchardanalysis for determination of binding affinity (see Scatchard, Ann. NYAcad. Sci. 51: 660-672, 1949) and calorimetric assays (Cunningham etal., Science 253:545-48, 1991; Cunningham et al., Science 245:821-25,1991).

[0169] Zcytor17 polypeptides can also be used to prepare antibodies thatbind to zcytor17 epitopes, peptides or polypeptides. The zcytor17polypeptide or a fragment thereof serves as an antigen (immunogen) toinoculate an animal and elicit an immune response. One of skill in theart would recognize that antigenic, epitope-bearing polypeptides containa sequence of at least 6, preferably at least 9, and more preferably atleast 15 to about 30 contiguous amino acid residues of a zcytor17polypeptide (e.g., SEQ ID NO:54, SEQ ID NO:57, and the like).Polypeptides comprising a larger portion of a zcytor17 polypeptide,i.e., from 30 to 100 residues up to the entire length of the amino acidsequence are included. Antigens or immunogenic epitopes can also includeattached tags, adjuvants and carriers, as described herein. Suitableantigens include the zcytor17 polypeptide encoded by SEQ ID NO:2 fromamino acid number 20 (Ala) to amino acid number 732 (Val), or acontiguous 9 to 713, or 30 or 50 to 713 amino acid fragment thereof; andSEQ ID NO:46 from amino acid number 20 (Ala) to amino acid number 649(Ile), or a contiguous 9 to 630, or 30 or 50 to 630 amino acid fragmentthereof; and SEQ ID NO:54 from amino acid number 33 (Ala) to amino acidnumber 662 (Ile), or a contiguous 9 to 630, or 30 or 50 to 630 aminoacid fragment thereof. Preferred peptides to use as antigens are theextracellular domain, cytokine binding domain, fibronectin type IIIdomain, intracellular signaling domain, Box I and Box II sites or otherdomains and motifs disclosed herein, or a combination thereof; andzcytor17 hydrophilic peptides such as those predicted by one of skill inthe art from a hydrophobicity plot, determined for example, from aHopp/Woods hydrophilicity profile based on a sliding six-residue window,with buried G, S, and T residues and exposed H, Y, and W residuesignored. Zcytor17 hydrophilic peptides include peptides comprising aminoacid sequences selected from the group consisting of: (1) amino acidresidues 43 through 48 of SEQ ID NO:2 and SEQ ID NO:46 (residues 56through 61 of SEQ ID NO:54); (2) amino acid residues 157 through 162 ofSEQ ID NO:2 and SEQ ID NO:46 (residues 170 through 175 of SEQ ID NO:54);(3) amino acid residues 158 through 163 of SEQ ID NO:2 and SEQ ID NO:46(171 through 176 of SEQ ID NO:54); (4) amino acid residues 221 through226 of SEQ ID NO:2 and SEQ ID NO:46 (234 through 239 of SEQ ID NO:54);and (5) amino acid residues 426 through 431 of SEQ ID NO:2 and SEQ IDNO:46 (residues 439 through 444 of SEQ ID NO:54). In addition,hydrophilic epitopes predicted from a Jameson-Wolf plot Jameson-Wolfplot, e.g., using DNASTAR Protean program (DNASTAR, Inc., Madison,Wis.), are also suitable antigens. In addition, conserved motifs, andvariable regions between conserved motifs of zcytor17 are suitableantigens. Antibodies generated from this immune response can be isolatedand purified as described herein. Methods for preparing and isolatingpolyclonal and monoclonal antibodies are well known in the art. See, forexample, Current Protocols in Immunology, Cooligan, et al. (eds.),National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrooket al., Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor, N.Y., 1989; and Hurrell, J. G. R., Ed., MonoclonalHybridoma Antibodies: Techniques and Applications, CRC Press, Inc., BocaRaton, Fla., 1982.

[0170] As would be evident to one of ordinary skill in the art,polyclonal antibodies can be generated from inoculating a variety ofwarm-blooded animals such as horses, cows, goats, sheep, dogs, chickens,rabbits, mice, and rats with a zcytor17 polypeptide or a fragmentthereof. The immunogenicity of a zcytor17 polypeptide may be increasedthrough the use of an adjuvant, such as alum (aluminum hydroxide) orFreund's complete or incomplete adjuvant. Polypeptides useful forimmunization also include fusion polypeptides, such as fusions ofzcytor17 or a portion thereof with an immunoglobulin polypeptide or withmaltose binding protein. The polypeptide immunogen may be a full-lengthmolecule or a portion thereof. If the polypeptide portion is“hapten-like”, such portion may be advantageously joined or linked to amacromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovineserum albumin (BSA) or tetanus toxoid) for immunization.

[0171] As used herein, the term “antibodies” includes polyclonalantibodies, affinity-purified polyclonal antibodies, monoclonalantibodies, and antigen-binding fragments, such as F(ab′)₂ and Fabproteolytic fragments. Genetically engineered intact antibodies orfragments, such as chimeric antibodies, Fv fragments, single chainantibodies and the like, as well as synthetic antigen-binding peptidesand polypeptides, are also included. Non-human antibodies may behumanized by grafting non-human CDRs onto human framework and constantregions, or by incorporating the entire non-human variable domains(optionally “cloaking” them with a human-like surface by replacement ofexposed residues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Moreover, human antibodies can beproduced in transgenic, non-human animals that have been engineered tocontain human immunoglobulin genes as disclosed in WIPO Publication WO98/24893. It is preferred that the endogenous immunoglobulin genes inthese animals be inactivated or eliminated, such as by homologousrecombination.

[0172] Alternative techniques for generating or selecting antibodiesuseful herein include in vitro exposure of lymphocytes to zcytor17protein or peptide, and selection of antibody display libraries in phageor similar vectors (for instance, through use of immobilized or labeledzcytor17 protein or peptide). Genes encoding polypeptides havingpotential zcytor17 polypeptide binding domains can be obtained byscreening random peptide libraries displayed on phage (phage display) oron bacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. These random peptidedisplay libraries can be used to screen for peptides which interact witha known target which can be a protein or polypeptide, such as a ligandor receptor, a biological or synthetic macromolecule, or organic orinorganic substances. Techniques for creating and screening such randompeptide display libraries are known in the art (Ladner et al., U.S. Pat.No. 5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al.,U.S. Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andrandom peptide display libraries and kits for screening such librariesare available commercially, for instance from Clontech (Palo Alto,Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.(Beverly, Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using thezcytor17 sequences disclosed herein to identify proteins which bind tozcytor17. These “binding peptides” which interact with zcytor17polypeptides can be used for tagging cells that express the receptor;for isolating homolog polypeptides by affinity purification; they can bedirectly or indirectly conjugated to drugs, toxins, radionuclides andthe like. These binding peptides can also be used in analytical methodssuch as for screening expression libraries and neutralizing activity.The binding peptides can also be used for diagnostic assays fordetermining circulating levels of zcytor17 polypeptides; for detectingor quantitating soluble zcytor17 polypeptides as marker of underlyingpathology or disease. These binding peptides can also act as zcytor17“antagonists” to block zcytor17 binding and signal transduction in vitroand in vivo. These anti-zcytor17 binding peptides would be useful forinhibiting the action of a ligand that binds with zcytor17.

[0173] Antibodies are considered to be specifically binding if: 1) theyexhibit a threshold level of binding activity, and 2) they do notsignificantly cross-react with related polypeptide molecules. Athreshold level of binding is determined if anti-zcytor17 antibodiesherein bind to a zcytor17 polypeptide, peptide or epitope with anaffinity at least 10-fold greater than the binding affinity to control(non-zcytor17) polypeptide. It is preferred that the antibodies exhibita binding affinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ orgreater, more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹or greater. The binding affinity of an antibody can be readilydetermined by one of ordinary skill in the art, for example, byScatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660-672,1949).

[0174] Whether anti-zcytor17 antibodies do not significantly cross-reactwith related polypeptide molecules is shown, for example, by theantibody detecting zcytor17 polypeptide but not known relatedpolypeptides using a standard Western blot analysis (Ausubel et al.,ibid.). Examples of known related polypeptides are those disclosed inthe prior art, such as known orthologs, and paralogs, and similar knownmembers of a protein family (e.g., gp130, LIF, WSX-1 and IL12receptors). Screening can also be done using non-human zcytor17, andzcytor17 mutant polypeptides. Moreover, antibodies can be “screenedagainst” known related polypeptides, to isolate a population thatspecifically binds to the zcytor17 polypeptides. For example, antibodiesraised to zcytor17 are adsorbed to related polypeptides adhered toinsoluble matrix; antibodies specific to zcytor17 will flow through thematrix under the proper buffer conditions. Screening allows isolation ofpolyclonal and monoclonal antibodies non-crossreactive to known closelyrelated polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane(eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols inImmunology, Cooligan, et al. (eds.), National Institutes of Health, JohnWiley and Sons, Inc., 1995). Screening and isolation of specificantibodies is well known in the art. See, Fundamental Immunology, Paul(eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98,1988; Monoclonal Antibodies: Principles and Practice, Goding, J. W.(eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol.2: 67-101, 1984. Specifically binding anti-zcytor17 antibodies can bedetected by a number of methods in the art, and disclosed below.

[0175] A variety of assays known to those skilled in the art can beutilized to detect antibodies which specifically bind to zcytor17proteins or peptides. Exemplary assays are described in detail inAntibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold SpringHarbor Laboratory Press, 1988. Representative examples of such assaysinclude: concurrent immunoelectrophoresis, radioimmunoassay,radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA),dot blot or Western blot assay, inhibition or competition assay, andsandwich assay. In addition, antibodies can be screened for binding towild-type versus mutant zcytor17 protein or polypeptide.

[0176] Antibodies to zcytor17 may be used for tagging cells that expresszcytor17, such as cells that naturally express zcytor17 such as monocyteand prostate cells, as well as cells that are transformed with zcytor17;for isolating zcytor17 by affinity purification; for diagnostic assaysfor determining circulating levels of zcytor17 polypeptides; fordetecting or quantitating soluble zcytor17 as marker of underlyingpathology or disease; in analytical methods employing FACS; forscreening expression libraries; for generating anti-idiotypicantibodies; and as neutralizing antibodies or as antagonists to blockzcytor17 activity in vitro and in vivo. Suitable direct tags or labelsinclude radionuclides, enzymes, substrates, cofactors, inhibitors,fluorescent markers, chemiluminescent markers, magnetic particles andthe like; indirect tags or labels may feature use of biotin-avidin orother complement/anti-complement pairs as intermediates. Antibodiesherein may also be directly or indirectly conjugated to drugs, toxins,radionuclides and the like, and these conjugates used for in vivodiagnostic or therapeutic applications. Moreover, antibodies to zcytor17or fragments thereof may be used in vitro to detect denatured zcytor17or fragments thereof in assays, for example, Western Blots or otherassays known in the art.

[0177] Antibodies to zcytor17 are useful for tagging cells that expressthe receptor and assaying Zcytor17 expression levels, for affinitypurification, within diagnostic assays for determining circulatinglevels of soluble receptor polypeptides, analytical methods employingfluorescence-activated cell sorting. Divalent antibodies may be used asagonists to mimic the effect of a zcytor17 ligand.

[0178] Antibodies herein can also be directly or indirectly conjugatedto drugs, toxins, radionuclides and the like, and these conjugates usedfor in vivo diagnostic or therapeutic applications. For instance,antibodies or binding polypeptides which recognize zcytor17 of thepresent invention can be used to identify or treat tissues or organsthat express a corresponding anti-complementary molecule (i.e., azcytor17 receptor). More specifically, anti-zcytor17 antibodies, orbioactive fragments or portions thereof, can be coupled to detectable orcytotoxic molecules and delivered to a mammal having cells, tissues ororgans that express the zcytor17 molecule.

[0179] Suitable detectable molecules may be directly or indirectlyattached to polypeptides that bind zcytor17 (“binding polypeptides,”including binding peptides disclosed above), antibodies, or bioactivefragments or portions thereof. Suitable detectable molecules includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmarkers, chemiluminescent markers, magnetic particles and the like.Suitable cytotoxic molecules may be directly or indirectly attached tothe polypeptide or antibody, and include bacterial or plant toxins (forinstance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and thelike), as well as therapeutic radionuclides, such as iodine-131,rhenium-188 or yttrium-90 (either directly attached to the polypeptideor antibody, or indirectly attached through means of a chelating moiety,for instance). Binding polypeptides or antibodies may also be conjugatedto cytotoxic drugs, such as adriamycin. For indirect attachment of adetectable or cytotoxic molecule, the detectable or cytotoxic moleculecan be conjugated with a member of a complementary/anticomplementarypair, where the other member is bound to the binding polypeptide orantibody portion. For these purposes, biotin/streptavidin is anexemplary complementary/anticomplementary pair.

[0180] In another embodiment, binding polypeptide-toxin fusion proteinsor antibody-toxin fusion proteins can be used for targeted cell ortissue inhibition or ablation (for instance, to treat cancer cells ortissues). Alternatively, if the binding polypeptide has multiplefunctional domains (i.e., an activation domain or a ligand bindingdomain, plus a targeting domain), a fusion protein including only thetargeting domain may be suitable for directing a detectable molecule, acytotoxic molecule or a complementary molecule to a cell or tissue typeof interest. In instances where the fusion protein including only asingle domain includes a complementary molecule, the anti-complementarymolecule can be conjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting vehicle for cell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

[0181] In another embodiment, zcytor17 binding polypeptide-cytokine orantibody-cytokine fusion proteins can be used for enhancing in vivokilling of target tissues (for example, blood, lymphoid, colon, and bonemarrow cancers), if the binding polypeptide-cytokine or anti-zcytor17antibody targets the hyperproliferative cell (See, generally, Hornick etal., Blood 89:4437-47, 1997). They described fusion proteins enabletargeting of a cytokine to a desired site of action, thereby providingan elevated local concentration of cytokine. Suitable anti-zcytor17antibodies target an undesirable cell or tissue (i.e., a tumor or aleukemia), and the fused cytokine mediates improved target cell lysis byeffector cells. Suitable cytokines for this purpose include interleukin2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), forinstance.

[0182] Alternatively, zcytor17 binding polypeptide or antibody fusionproteins described herein can be used for enhancing in vivo killing oftarget tissues by directly stimulating a zcytor17-modulated apoptoticpathway, resulting in cell death of hyperproliferative cells expressingzcytor17.

[0183] The bioactive binding polypeptide or antibody conjugatesdescribed herein can be delivered orally, intravenously, intraarteriallyor intraductally, or may be introduced locally at the intended site ofaction.

[0184] Four-helix bundle cytokines that bind to cytokine receptors aswell as other proteins produced by activated lymphocytes play animportant biological role in cell differentiation, activation,recruitment and homeostasis of cells throughout the body. Therapeuticutility includes treatment of diseases which require immune regulationincluding autoimmune diseases, such as, rheumatoid arthritis, multiplesclerosis, myasthenia gravis, systemic lupus erythomatosis and diabetes.Zcytor17 receptor antagonists or agonists, including soluble receptors,anti-receptor antibodies, and the natural ligand, may be important inthe regulation of inflammation, and therefore would be useful intreating rheumatoid arthritis, asthma, ulcerative colitis, inflammatorybowel disease, Crohn's disease, and sepsis. There may be a role ofzcytor17 antagonists or agonists, including soluble receptors,anti-receptor antibodies and the natural ligand, in mediatingtumorgenesis, and therefore would be useful in the treatment of cancer.Zcytor17 antagonists or agonists, including soluble receptors,anti-receptor antibodies and the natural ligand, may be a potentialtherapeutic in suppressing the immune system which would be importantfor reducing graft rejection or in prevention of graft vs. host disease.

[0185] Alternatively, zcytor17 antagonists or agonists, includingsoluble receptors, anti-zcytor17 receptor antibodies and the naturalligand may activate the immune system which would be important inboosting immunity to infectious diseases, treating immunocompromisedpatients, such as HIV+ patient, or in improving vaccines. In particular,zcytor17 antagonists or agonists, including soluble receptors and thenatural ligand can modulate, stimulate or expand NK cells, or theirprogenitors, and would provide therapeutic value in treatment of viralinfection, and as an anti-neoplastic factor. NK cells are thought toplay a major role in elimination of metastatic tumor cells and patientswith both metastases and solid tumors have decreased levels of NK cellactivity (Whiteside et. al., Curr. Top. Microbiol. Immunol. 230:221-244,1998).

[0186] Polynucleotides encoding zcytor17 polypeptides are useful withingene therapy applications where it is desired to increase or inhibitzcytor17 activity. If a mammal has a mutated or absent zcytor17 gene,the zcytor17 gene can be introduced into the cells of the mammal. In oneembodiment, a gene encoding a zcytor17 polypeptide is introduced in vivoin a viral vector. Such vectors include an attenuated or defective DNAvirus, such as, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. A defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Examples ofparticular vectors include, but are not limited to, a defective herpessimplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.2:320-30, 1991); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30,1992; and a defective adeno-associated virus vector (Samulski et al., J.Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

[0187] In another embodiment, a zcytor17 gene can be introduced in aretroviral vector, e.g., as described in Anderson et al., U.S. Pat. No.5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263;International Pat. Publication No. WO 95/07358, published Mar. 16, 1995by Dougherty et al.; and Kuo et al., Blood 82:845, 1993. Alternatively,the vector can be introduced by lipofection in vivo using liposomes.Synthetic cationic lipids can be used to prepare liposomes for in vivotransfection of a gene encoding a marker (Felgner et al., Proc. Natl.Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci.USA 85:8027-31, 1988). The use of lipofection to introduce exogenousgenes into specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. More particularly, directing transfection to particularcells represents one area of benefit. For instance, directingtransfection to particular cell types would be particularly advantageousin a tissue with cellular heterogeneity, such as the pancreas, liver,kidney, and brain. Lipids may be chemically coupled to other moleculesfor the purpose of targeting. Targeted peptides (e.g., hormones orneurotransmitters), proteins such as antibodies, or non-peptidemolecules can be coupled to liposomes chemically.

[0188] It is possible to remove the target cells from the body; tointroduce the vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

[0189] Antisense methodology can be used to inhibit zcytor17 genetranscription, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of azcytor17-encoding polynucleotide (e.g., a polynucleotide as set froth inSEQ ID NO:1, SEQ ID NO:45, SEQ ID NO:53, or SEQ ID NO:57) are designedto bind to zcytor17-encoding mRNA and to inhibit translation of suchmRNA. Such antisense polynucleotides are used to inhibit expression ofzcytor17 polypeptide-encoding genes in cell culture or in a subject.

[0190] In addition, as a cell surface molecule, zcytor17 polypeptidescan be used as a target to introduce gene therapy into a cell. Thisapplication would be particularly appropriate for introducingtherapeutic genes into cells in which zcytor17 is normally expressed,such as lymphoid tissue, bone marrow, prostate, thyroid, monocytes andPBLs, or cancer cells which express zcytor17 polypeptide. For example,viral gene therapy, such as described above, can be targeted to specificcell types in which express a cellular receptor, such as zcytor17polypeptide, rather than the viral receptor. Antibodies, or othermolecules that recognize zcytor17 molecules on the target cell's surfacecan be used to direct the virus to infect and administer genetherapeutic material to that target cell. See, Woo, S. L. C, NatureBiotech. 14:1538, 1996; Wickham, T. J. et al, Nature Biotech.14:1570-1573, 1996; Douglas, J. T et al., Nature Biotech. 14:1574-1578,1996; Rihova, B., Crit. Rev. Biotechnol. 17:149-169, 1997; and Vile, R.G. et al., Mol. Med. Today 4:84-92, 1998. For example, a bispecificantibody containing a virus-neutralizing Fab fragment coupled to azcytor17-specific antibody can be used to direct the virus to cellsexpressing the zcytor17 receptor and allow efficient entry of the viruscontaining a genetic element into the cells. See, for example, Wickham,T. J., et al., J. Virol. 71:7663-7669, 1997; and Wickham, T. J., et al.,J. Virol. 70:6831-6838, 1996.

[0191] Moreover, anti-zcytor17 antibodies and binding fragments can beused for tagging and sorting cells that specifically-express Zcytor17,such as mononuclear cells, lymphoid cells, e.g, non-activated andactivated monocyte cells, such as activated CD3+, CD4+ and CD8+ cells,CD19+B-cells, and other cells, described herein. Such methods of celltagging and sorting are well known in the art (see, e.g., “MolecularBiology of the Cell”, 3^(rd) Ed., Albert, B. et al. (Garland Publishing,London & New York, 1994). One of skill in the art would recognize theimportance of separating cell tissue types to study cells, and the useof antibodies to separate specific cell tissue types. Basically,antibodies that bind to the surface of a cell type are coupled tovarious matrices such as collagen, polysaccharide beads, or plastic toform an affinity surface to which only cells recognized by theantibodies will adhere. The bound cells are then recovered byconventional techniques. Other methods involve separating cells by afluorescence-activated cell sorter (FACS). In this technique one labelscells with antibodies that are coupled to a fluorescent dye. The labeledcells are then separated from unlabeled cells in a FACS machine. In FACSsorting individual cells traveling in single file pass through a laserbeam and the fluorescence of each cell is measured. Slightly furtherdown-stream, tiny droplets, most containing either one or no cells, areformed by a vibrating nozzle. The droplets containing a single cell areautomatically give a positive or negative charge at the moment offormation, depending on whether the cell they contain is fluorescent,and then deflected by a strong electric field into an appropriatecontainer. Such machines can select 1 cell in 1000 and sort about 5000cells each second. This produces a uniform population of cells for cellculture.

[0192] One of skill in the art would recognize that the antibodies tothe Zcytor17 polypeptides of the present invention are useful, becausenot all tissue types express the Zcytor17 receptor and because it isimportant that biologists be able to separate specific cell types forfurther study and/or therapeutic re-implantation into the body. This isparticularly relevant in cells such as immune cells, wherein zcytor17 isexpressed.

[0193] The present invention also provides reagents that will find usein diagnostic applications. For example, the zcytor17 gene, a probecomprising zcytor17 DNA or RNA or a subsequence thereof can be used todetermine if the zcytor17 gene is present on chromosome 5 or if amutation has occurred. Zcytor17 is located at the 5q11 region ofchromosome 5 (See, Example 4). Detectable chromosomal aberrations at thezcytor17 gene locus include, but are not limited to, aneuploidy, genecopy number changes, loss of heterogeneity (LOH), translocations,insertions, deletions, restriction site changes and rearrangements. Suchaberrations can be detected using polynucleotides of the presentinvention by employing molecular genetic techniques, such as restrictionfragment length polymorphism (RFLP) analysis, fluorescence in situhybridization methods, short tandem repeat (STR) analysis employing PCRtechniques, and other genetic linkage analysis techniques known in theart (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest108:255-65, 1995).

[0194] The precise knowledge of a gene's position can be useful for anumber of purposes, including: 1) determining if a sequence is part ofan existing contig and obtaining additional surrounding geneticsequences in various forms, such as YACs, BACs or cDNA clones; 2)providing a possible candidate gene for an inheritable disease whichshows linkage to the same chromosomal region; and 3) cross-referencingmodel organisms, such as mouse, which may aid in determining whatfunction a particular gene might have.

[0195] The zcytor17 gene is located at the 5q11 region of chromosome 5.Several genes of known function map to this region. For example, aclosely related class I cytokine receptor, gp130, also maps chromosome5q11 suggesting that the 5q11 region is an important region for cytokinereceptor expression. Zcytor17 maps in the 5q11 chromosomal region (firstregion distal of the centromere on the q-arm) and gp130 appears to beabout 920.7 kb distal of Zcytor 17. Moreover, the closely related classI cytokine receptor LIFR maps just on the other side of the centromereon the p-arm in the 5p13-p12 region. The gp130 cytokine receptor isshared by several other cytokine receptors to form heterodimericcomplexes, that enable signaling by cytokines such as IL-6, leukemiainhibitory factor (LIF), oncostatin M (OSM), and ciliary neurotropicfactor (CNTF). Moreover, gp130 may form a heterodimeric, trimeric (e.g.,with gp130+ LIF receptor), or multimeric complex with the zcytor17polypeptide in order to signal. Moreover, as discussed herein, cytokinereceptors such as zcytor17 and gp130 play important roles in immune cellfunction, proliferation, migration, inflammation and the like. As such,zcytor17 polynucleotides, polypeptides, and anti-zcytor17 antibodiesserve an important use as a diagnostic to detect defects in the zcytor17gene or protein, or defects in surrounding chromosomal regions at the5q11 region of chromosome 5.

[0196] Moreover, several disease-related genes cluster in the 5q11region that are associated with human disorders. One of skill in the artwould recognize that a marker in 5q11 such as the zcytor17polynucleotides of the present invention, would be useful in detectingchromosomal aberrations associated with human disease, since aberrationsin and around 5q11 are known to be linked to human disease. For example,5q11-q13.3 duplications, partial trisomy, and translocations, areassociated with multiple anomalies including schizophrenia, a commonpsychosis. In addition, Maroteaux-Lamy Syndrome, or mucopolysaccaridosistypes VI (5q11-q13) and Klippel-Feil syndrome (5q11.2) are associatedwith translocation at this locus. In addition, these diseases are linkedto large chromosomal rearrangements, such as chromosome duplication,translocation or loss of heterogeneity in the 5q11 region chromosome 5.Using, for example, polynucleotides of the present invention inconjunction with known methods in the art described herein, suchrearrangements at or around 5q11 can be detected. Moreover, amongstother genetic loci, those for split-hand/foot malformation, type 1(SHFM1) (5q), Sandhoff disease (5q13), glucocorticoid receptor (5q31),dihydrofolate reductase (DHFR) (5q11.2-q13.2) spinal muscular atrophy(5q12.2-q13.3) and Pituitary Hormone Deficiency (5q) all manifestthemselves in human disease states as well as map to this region of thehuman genome. See the Online Mendellian Inheritance of Man (OMIM™,National Center for Biotechnology Information, National Library ofMedicine. Bethesda, Md.) gene map, and references therein, for thisregion of chromosome 5 on a publicly available WWW server(http://www3.ncbi.nlm.nih.gov/htbin-post/Omim/getmap?chromosome=5q11 andsurrounding loci). All of these serve as possible candidate genes for aninheritable disease that show linkage to the same chromosomal region asthe zcytor17 gene.

[0197] Similarly, defects in the zcytor17 locus itself may result in aheritable human disease states as discussed herein. One of skill in theart would appreciate that defects in cytokine receptors are known tocause disease states in humans. For example, growth hormone receptormutation results in dwarfism (Amselem, S et al., New Eng. J. Med. 321:989-995, 1989), IL-2 receptor gamma mutation results in severe combinedimmunodeficiency (SCID) (Noguchi, M et al., Cell 73: 147-157, 1993),c-Mpl mutation results in thrombocytopenia (Ihara, K et al., Proc. Nat.Acad. Sci. 96: 3132-3136, 1999), and severe mycobacterial and Salmonellainfections result in interleukin-12 receptor-deficient patients (deJong, R et al., Science 280: 1435-1438, 1998), amongst others. Thus,similarly, defects in zcytor17 can cause a disease state orsusceptibility to disease or infection. As the zcytor17 gene is locatedat the 5q11 region zcytor17, polynucleotide probes can be used to detectchromosome 5q11 loss, trisomy, duplication or translocation associatedwith human diseases, such as immune cell cancers, bone marrow cancers,prostate cancer, thyroid, parathyroid or other cancers, or immunediseases. Moreover, molecules of the present invention, such as thepolypeptides, antagonists, agonists, polynucleotides and antibodies ofthe present invention would aid in the detection, diagnosis prevention,and treatment associated with a zcytor17 genetic defect.

[0198] Molecules of the present invention, such as the polypeptides,antagonists, agonists, polynucleotides and antibodies of the presentinvention would aid in the detection, diagnosis prevention, andtreatment associated with a zcytor17 genetic defect.

[0199] A diagnostic could assist physicians in determining the type ofdisease and appropriate associated therapy, or assistance in geneticcounseling. As such, the inventive anti-zcytor17 antibodies,polynucleotides, and polypeptides can be used for the detection ofzcytor17 polypeptide, mRNA or anti-zcytor17 antibodies, thus serving asmarkers and be directly used for detecting or genetic diseases orcancers, as described herein, using methods known in the art anddescribed herein. Further, zcytor17 polynucleotide probes can be used todetect abnormalities or genotypes associated with chromosome 5q11deletions and translocations associated with human diseases, othertranslocations involved with malignant progression of tumors or other5q11 mutations, which are expected to be involved in chromosomerearrangements in malignancy; or in other cancers, or in spontaneousabortion. Similarly, zcytor17 polynucleotide probes can be used todetect abnormalities or genotypes associated with chromosome 5q11trisomy and chromosome loss associated with human diseases orspontaneous abortion. Thus, zcytor17 polynucleotide probes can be usedto detect abnormalities or genotypes associated with these defects.

[0200] As discussed above, defects in the zcytor17 gene itself mayresult in a heritable human disease state. Molecules of the presentinvention, such as the polypeptides, antagonists, agonists,polynucleotides and antibodies of the present invention would aid in thedetection, diagnosis prevention, and treatment associated with azcytor17 genetic defect. In addition, zcytor17 polynucleotide probes canbe used to detect allelic differences between diseased or non-diseasedindividuals at the zcytor17 chromosomal locus. As such, the zcytor17sequences can be used as diagnostics in forensic DNA profiling.

[0201] In general, the diagnostic methods used in genetic linkageanalysis, to detect a genetic abnormality or aberration in a patient,are known in the art. Analytical probes will be generally at least 20 ntin length, although somewhat shorter probes can be used (e.g., 14-17nt). PCR primers are at least 5 nt in length, preferably 15 or more,more preferably 20-30 nt. For gross analysis of genes, or chromosomalDNA, a zcytor17 polynucleotide probe may comprise an entire exon ormore. Exons are readily determined by one of skill in the art bycomparing zcytor17 sequences (e.g., SEQ ID NO:54) with the human genomicDNA for zcytor17 (Genbank Accession No. AQ002781). In general, thediagnostic methods used in genetic linkage analysis, to detect a geneticabnormality or aberration in a patient, are known in the art. Mostdiagnostic methods comprise the steps of (a) obtaining a genetic samplefrom a potentially diseased patient, diseased patient or potentialnon-diseased carrier of a recessive disease allele; (b) producing afirst reaction product by incubating the genetic sample with a zcytor17polynucleotide probe wherein the polynucleotide will hybridize tocomplementary polynucleotide sequence, such as in RFLP analysis or byincubating the genetic sample with sense and antisense primers in a PCRreaction under appropriate PCR reaction conditions; (iii) Visualizingthe first reaction product by gel electrophoresis and/or other knownmethod such as visualizing the first reaction product with a zcytor17polynucleotide probe wherein the polynucleotide will hybridize to thecomplementary polynucleotide sequence of the first reaction; and (iv)comparing the visualized first reaction product to a second controlreaction product of a genetic sample from wild type patient. Adifference between the first reaction product and the control reactionproduct is indicative of a genetic abnormality in the diseased orpotentially diseased patient, or the presence of a heterozygousrecessive carrier phenotype for a non-diseased patient, or the presenceof a genetic defect in a tumor from a diseased patient, or the presenceof a genetic abnormality in a fetus or pre-implantation embryo. Forexample, a difference in restriction fragment pattern, length of PCRproducts, length of repetitive sequences at the zcytor17 genetic locus,and the like, are indicative of a genetic abnormality, geneticaberration, or allelic difference in comparison to the normal wild typecontrol. Controls can be from unaffected family members, or unrelatedindividuals, depending on the test and availability of samples. Geneticsamples for use within the present invention include genomic DNA, mRNA,and cDNA isolated form any tissue or other biological sample from apatient, such as but not limited to, blood, saliva, semen, embryoniccells, amniotic fluid, and the like. The polynucleotide probe or primercan be RNA or DNA, and will comprise a portion of SEQ ID NO:1, SEQ IDNO:45 or SEQ ID NO:53, the complement of SEQ ID NO:1, SEQ ID NO:45 orSEQ ID NO:53, or an RNA equivalent thereof. Such methods of showinggenetic linkage analysis to human disease phenotypes are well known inthe art. For reference to PCR based methods in diagnostics see see,generally, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), White (ed.), PCR Protocols: Current Methods andApplications (Humana Press, Inc. 1993), Cotter (ed.), MolecularDiagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek(eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.),Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer(ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).

[0202] Mutations associated with the zcytor17 locus can be detectedusing nucleic acid molecules of the present invention by employingstandard methods for direct mutation analysis, such as restrictionfragment length polymorphism analysis, short tandem repeat analysisemploying PCR techniques, amplification-refractory mutation systemanalysis, single-strand conformation polymorphism detection, RNasecleavage methods, denaturing gradient gel electrophoresis,fluorescence-assisted mismatch analysis, and other genetic analysistechniques known in the art (see, for example, Mathew (ed.), Protocolsin Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest108:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (HumanPress, Inc. 1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases(Humana Press, Inc. 1996), Landegren (ed.), Laboratory Protocols forMutation Detection (Oxford University Press 1996), Birren et al. (eds.),Genome Analysis, Vol. 2: Detecting Genes (Cold Spring Harbor LaboratoryPress 1998), Dracopoli et al. (eds.), Current Protocols in HumanGenetics (John Wiley & Sons 1998), and Richards and Ward, “MolecularDiagnostic Testing,” in Principles of Molecular Medicine, pages 83-88(Humana Press, Inc. 1998)). Direct analysis of an zcytor17 gene for amutation can be performed using a subject's genomic DNA. Methods foramplifying genomic DNA, obtained for example from peripheral bloodlymphocytes, are well-known to those of skill in the art (see, forexample, Dracopoli et al. (eds.), Current Protocols in Human Genetics,at pages 7.1.6 to 7.1.7 (John Wiley & Sons 1998)).

[0203] Mice engineered to express the zcytor17 gene, referred to as“transgenic mice,” and mice that exhibit a complete absence of zcytor17gene function, referred to as “knockout mice,” may also be generated(Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature366:740-42, 1993; Capecchi, M. R., Science 244: 1288-1292, 1989;Palmiter, R. D. et al. Annu Rev Genet. 20: 465-499, 1986). For example,transgenic mice that over-express zcytor17, either ubiquitously or undera tissue-specific or tissue-restricted promoter can be used to askwhether over-expression causes a phenotype. For example, over-expressionof a wild-type zcytor17 polypeptide, polypeptide fragment or a mutantthereof may alter normal cellular processes, resulting in a phenotypethat identifies a tissue in which zcytor17 expression is functionallyrelevant and may indicate a therapeutic target for the zcytor17, itsagonists or antagonists. For example, a preferred transgenic mouse toengineer is one that expresses a “dominant-negative” phenotype, such asone that over-expresses the zcytor17 polypeptide comprising anextracellular cytokine binding domain with the transmembrane domainattached (approximately amino acids 20 (Ala) to 543 (Leu) of SEQ ID NO:2and SEQ ID NO:46; or 33 (Ala) to 556 (Leu) of SEQ ID NO:54). Anotherpreferred transgenic mouse is one that over-expresses zcytor17 solublereceptors, such as those disclosed herein. Moreover, suchover-expression may result in a phenotype that shows similarity withhuman diseases. Similarly, knockout zcytor17 mice can be used todetermine where zcytor17 is absolutely required in vivo. The phenotypeof knockout mice is predictive of the in vivo effects of a zcytor17antagonist, such as those described herein, may have. The mouse zcytor17mRNA, cDNA (SEQ ID NO:56 and/or SEQ ID NO:92) and genomic DNA, are usedto generate knockout mice. These transgenic and knockout mice may beemployed to study the zcytor17 gene and the protein encoded thereby inan in vivo system, and can be used as in vivo models for correspondinghuman or animal diseases (such as those in commercially viable animalpopulations). The mouse models of the present invention are particularlyrelevant as, immune system models, inflammation or tumor models for thestudy of cancer biology and progression. Such models are useful in thedevelopment and efficacy of therapeutic molecules used in human immunediseases, inflammation and cancers. Because increases in zcytor17expression, as well as decreases in zcytor17 expression are associatedwith monocytes, monocyte activation, and prostate cells, and may beassociated with inflammation and cancers, both transgenic mice andknockout mice would serve as useful animal models for human disease.Moreover, in a preferred embodiment, zcytor17 transgenic mouse can serveas an animal model for specific diseases, particularly those associatedwith monocytes. Moreover, transgenic mice expression of zcytor17antisense polynucleotides or ribozymes directed against zcytor17,described herein, can be used analogously to transgenic mice describedabove.

[0204] For pharmaceutical use, the soluble receptor polypeptides of thepresent invention are formulated for parenteral, particularlyintravenous or subcutaneous, delivery according to conventional methods.Intravenous administration will be by bolus injection or infusion over atypical period of one to several hours. In general, pharmaceuticalformulations will include a zcytor17 soluble receptor polypeptide incombination with a pharmaceutically acceptable vehicle, such as saline,buffered saline, 5% dextrose in water or the like. Formulations mayfurther include one or more excipients, preservatives, solubilizers,buffering agents, albumin to prevent protein loss on vial surfaces, etc.Methods of formulation are well known in the art and are disclosed, forexample, in Remington: The Science and Practice of Pharmacy, Gennaro,ed., Mack Publishing Co., Easton, Pa., 19th ed., 1995. Therapeutic doseswill generally be in the range of 0.1 to 100 μg/kg of patient weight perday, preferably 0.5-20 mg/kg per day, with the exact dose determined bythe clinician according to accepted standards, taking into account thenature and severity of the condition to be treated, patient traits, etc.Determination of dose is within the level of ordinary skill in the art.The proteins may be administered for acute treatment, over one week orless, often over a period of one to three days or may be used in chronictreatment, over several months or years. In general, a therapeuticallyeffective amount of zcytor17 soluble receptor polypeptide is an amountsufficient to produce a clinically significant effect.

[0205] Polynucleotides and polypeptides of the present invention willadditionally find use as educational tools as a laboratory practicumkits for courses related to genetics and molecular biology, proteinchemistry and antibody production and analysis. Due to its uniquepolynucleotide and polypeptide sequence molecules of zcytor17 can beused as standards or as “unknowns” for testing purposes. For example,zcytor17 polynucleotides can be used as an aid, such as, for example, toteach a student how to prepare expression constructs for bacterial,viral, and/or mammalian expression, including fusion constructs, whereinzcytor17 is the gene to be expressed; for determining the restrictionendonuclease cleavage sites of the polynucleotides; determining mRNA andDNA localization of zcytor17 polynucleotides in tissues (i.e., byNorthern and Southern blotting as well as polymerase chain reaction);and for identifying related polynucleotides and polypeptides by nucleicacid hybridization.

[0206] Zcytor17 polypeptides can be used educationally as an aid toteach preparation of antibodies; identifying proteins by Westernblotting; protein purification; determining the weight of expressedzcytor17 polypeptides as a ratio to total protein expressed; identifyingpeptide cleavage sites; coupling amino and carboxyl terminal tags; aminoacid sequence analysis, as well as, but not limited to monitoringbiological activities of both the native and tagged protein (i.e.,receptor binding, signal transduction, proliferation, anddifferentiation) in vitro and in vivo. Zcytor17 polypeptides can also beused to teach analytical skills such as mass spectrometry, circulardichroism to determine conformation, especially of the four alphahelices, x-ray crystallography to determine the three-dimensionalstructure in atomic detail, nuclear magnetic resonance spectroscopy toreveal the structure of proteins in solution. For example, a kitcontaining the zcytor17 can be given to the student to analyze. Sincethe amino acid sequence would be known by the professor, the specificprotein can be given to the student as a test to determine the skills ordevelop the skills of the student, the teacher would then know whetheror not the student has correctly analyzed the polypeptide. Since everypolypeptide is unique, the educational utility of zcytor17 would beunique unto itself.

[0207] Moreover, since zcytor17 has a tissue-specific expression and isa polypeptide with a class I cytokine receptor structure and a distinctchromosomal localization, and expression pattern, activity can bemeasured using proliferation assays; luciferase and binding assaysdescribed herein. Moreover, expression of zcytor17 polynucleotides andpolypeptides in monocyte, prostate, lymphoid and other tissues can beanalyzed in order to train students in the use of diagnostic andtissue-specific identification and methods. Moreover zcytor17polynucleotides can be used to train students on the use of chromosomaldetection and diagnostic methods, since it's locus is known. Moreover,students can be specifically trained and educated about human chromosome1, and more specifically the locus 5q11 wherein the zcytor17 gene islocalized. Such assays are well known in the art, and can be used in aneducational setting to teach students about cytokine receptor proteinsand examine different properties, such as cellular effects on cells,enzyme kinetics, varying antibody binding affinities, tissuespecificity, and the like, between zcytor17 and other cytokine receptorpolypeptides in the art.

[0208] The antibodies which bind specifically to zcytor17 can be used asa teaching aid to instruct students how to prepare affinitychromatography columns to purify zcytor17, cloning and sequencing thepolynucleotide that encodes an antibody and thus as a practicum forteaching a student how to design humanized antibodies. Moreover,antibodies that bind specifically to zcytor17 can be used as a teachingaid for use in detection e.g., of activated monocyte cells, cellsorting, or lymphoid and prostate cancer tissue using histological, andin situ methods amongst others known in the art. The zcytor17 gene,polypeptide or antibody would then be packaged by reagent companies andsold to universities and other educational entities so that the studentsgain skill in art of molecular biology. Because each gene and protein isunique, each gene and protein creates unique challenges and learningexperiences for students in a lab practicum. Such educational kitscontaining the zcytor17 gene, polypeptide or antibody are consideredwithin the scope of the present invention.

[0209] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1

[0210] Identification and Isolation of Full-length Human zcytor17 cDNA

[0211] Zcytor18 was identified as a predicted full-length cDNA fromhuman genomic DNA. The sequence of the predicted full length zcytor17polynucleotide is shown in SEQ ID NO:1 and the corresponding polypeptideis shown in SEQ ID NO:2. To obtain a full-length cDNA from a tissuesource, 5′ and 3′ RACE were employed. Several oligonucleotide primerswere designed from the identified genomic sequence AQ002781 (Genbank).The primers were used for priming internally within the genomic sequenceto ultimately isolate a full-length cDNA.

[0212] A. 5′ RACE for zcytor17

[0213] A 5′ RACE product was generated using an HPVS cDNA library as atemplate and oligonucleotides ZC12,701 (SEQ ID NO:5) and ZC27,898 (SEQID NO:6) as primers. HPVS is an in-house cDNA library generated from ahuman prostate epithelial cell line (ATCC No. CRL-2221). The PCRreaction used approximately 1 μg of of plasmid DNA prepared from thecDNA library as a template, 5 μl of 10×PCR buffer (GIBCO/BRL), 5 μl of10 mM dNTPs (Perkin Elmer), 20 pmol each oligonucleotide, and 1 μl (5.0units) Taq polymerase (GIBCO/BRL) in a 50 ul reaction volume. Thisfirst-round 5′ RACE PCR reaction was run as follows: 30 cycles at 94° C.for 1 minute, 65° C. for 1 minute, 72° C. for 2 minutes, then 72° C. for7 minutes; 4° C. soak. An aliquot of 5′ RACE PCR product was removed andanalyzed on a 1.0% agarose gel. Multiple bands were seen on the gel.

[0214] The remaining 5′ RACE PCR product was ethanol precipitated anddiluted 1:50. A second-round nested 5′ RACE PCR reaction was run toamplify template cDNA sequence. This PCR reaction used oligonucleotidesZC14,063 (SEQ ID NO:7) and ZC27,899 (SEQ ID NO:8), which were designedto anneal to sequence internal of ZC12,701 (SEQ ID NO:5) and ZC27,898(SEQ ID NO:6). This nested PCR reaction was run as per the first-round5′ RACE reaction disclosed above. The resulting DNA products wereelectrophoresed on a 1.0% agarose gel and a prominent band atapproximately 900 bp was seen. The DNA band was gel purified andsequenced using standard methods. Sequence analyses revealed that theDNA product included part of the genomic AQ002781 DNA sequence (Genbank)and appeared to extend the cDNA sequence for zcytor17 on the 5′ end toinclude a translation initiating methionine residue and some 5′untranslated sequence. The polynucleotide sequence of the 5′ RACEproduct is shown in SEQ ID NO:9.

[0215] B. 3′ RACE for zcytor17

[0216] Primers for 3′ RACE were designed using the 5′ RACE product (SEQID NO:9) obtained above. A 3′ RACE product was generated using a humanprostate in-house cDNA library as a template and oligonucleotidesZC28,481 (SEQ ID NO:10) and ZC6,346 (SEQ ID NO:11) as primers. Thisfirst-round 3′ RACE PCR reaction was run under the conditions describedin Example 1A. An aliquot of 3′ RACE PCR product was removed andanalyzed on a 1.0% agarose gel. Multiple bands were seen on the gel.

[0217] The remaining 3′ RACE PCR product was ethanol precipitated anddiluted 1:40. A second-round nested 3′ RACE PCR reaction was run toamplify template cDNA sequence. This PCR reaction used oligonucleotidesZC28,480 (SEQ ID NO:12) and ZC26,405 (SEQ ID NO:13), which were designedto anneal to sequence internal of ZC28,481 (SEQ ID NO:10) and ZC6,346(SEQ ID NO:11). This nested PCR reaction was run as disclosed above. Theresulting DNA products were electrophoresed on a 1.0% agarose gel and aprominent band at approximately 2100 bp was seen.

[0218] The remaining DNA was ethanol precipitated and diluted 1:40. Athird-round nested 3′ RACE PCR reaction was run to amplify template cDNAsequence. This PCR reaction used oligonucleotides ZC27,895 (SEQ IDNO:14) and ZC5,020 (SEQ ID NO:15), which were designed to anneal tosequence internal of ZC28,480 (SEQ ID NO:12) and ZC26,405 (SEQ IDNO:13). This nested PCR reaction was run as disclosed above. Theresulting PCR products were electrophoresed on a 1.0% agarose gel and aprominent band at approximately 2000 bp was seen. The DNA band was gelpurified and sequenced. Sequence analyses revealed that the DNA productincluded part of the 5′ RACE product (SEQ ID NO:9) and appeared toextend the cDNA sequence for zcytor17 on the 3′ end to include atranslation stop codon and some 3′ untranslated sequence. Thepolynucleotide sequence of the 3′ RACE product is shown in SEQ ID NO:16.The polynucleotide sequence of the full-length zcytor17 is shown in SEQID NO:45 and the corresponding polypeptide sequence is shown in SEQ IDNO:46.

[0219] C. A Second 5′ RACE for zcytor17 Identified an AlternativeFull-length zcytor17

[0220] A 5′ RACE product was generated using a WI-38 cDNA library as atemplate and oligonucleotides ZC12,701 (SEQ ID NO:5) and ZC27,899 (SEQID NO:8) as primers. WI-28 is an in-house cDNA library generated from ahuman embryonic lung cell line (ATCC No. CRL-75). The PCR reaction usedapproximately 1 μg of of plasmid DNA prepared from the cDNA library as atemplate, 5 μl of 10×PCR buffer (GIBCO/BRL), 5 μl of 10 mM dNTPs (PerkinElmer), 20 pmol each oligonucleotide, and 1 μl (5.0 units) Taqpolymerase (GIBCO/BRL) in a 50 μl reaction volume. This first-round 5′RACE PCR reaction was run as follows: 30 cycles at 94° C. for 1 minute,65° C. for 1 minute, 72° C. for 2 minutes, then 72° C. for 7 minutes; 4°C. soak. An aliquot of 5′ RACE PCR product was removed and analyzed on a1.0% agarose gel. Multiple bands were seen on the gel.

[0221] The remaining DNA was ethanol precipitated and diluted 1:50. Asecond-round nested 5′ RACE PCR reaction was run to amplify templatecDNA sequence. This PCR reaction used oligonucleotides ZC14,063 (SEQ IDNO:25), and ZC27,900 (SEQ ID NO:51), which were designed to anneal tosequence internal of ZC12,701 (SEQ ID NO:5) and ZC27,899 (SEQ ID NO:8).This nested PCR reaction was run as per the first-round 5′ RACE reactiondisclosed above. The resulting DNA products were electrophoresed on a1.0% agarose gel and a prominent band at approximately 1200 bp was seen.The DNA band was gel purified and sequenced. Sequence analyses revealedthat the DNA product included part of the genomic DNA sequence AQ002781(Genbank) and appeared to extend the cDNA sequence for zcytor17 on the5′ end to include a translation initiating methionine residue and some5′ untranslated sequence. DNA sequencing showed that the polypeptidegenerated from translation from this alternative initiating methionine(shown in SEQ ID NO:53 at nucleotide 497) generates a second full-lengthform of zcytor17 that differs by an additional 13 amino acids in-frameat the N-terminus (MKLSPQPSCVNLG; SEQ ID NO:52) from that shown in SEQID NO:46. The polynucleotide sequence of the second full-length form ofzcytor17 is shown in SEQ ID NO:53 and the corresponding polypeptidesequence is shown in SEQ ID NO:54. The second full-length form ofzcytor17 (SEQ ID NO:53 and SEQ ID NO:54) is likely the most commonlyexpressed form.

Example 2

[0222] Identification and Isolation of Truncated Forms Human zcytor17cDNA

[0223] A. Isolation of a cDNA Coding for a Variant Form of zcytor17Truncated at the Fibronectin Domain

[0224] A 3′ RACE product for a truncated soluble form of zcytor17 wasgenerated using a protocol identical to that described above for 3′ RACE(Example 1B), except that the starting material was the HPVS cDNAlibrary. HPVS is an in-house cDNA library generated from a humanprostate epithelial cell line (ATCC No. CRL-2221). The resultingproducts from the third round nested 3′ RACE were electrophoresed on a1.0% agarose gel and a prominent band at approximately 700 bp was seen.The DNA band was gel purified and sequenced. Sequence analyses revealedthat the DNA product included part of the 5′ RACE product (SEQ ID NO:9)and appeared to extend the cDNA sequence for zcytor17 to include atranslation stop codon near the end of the cytokine-binding domain. Thiscould represent an expressed soluble form of the receptor truncatedwithin the fibronectin domain. The polynucleotide sequence of thesoluble form of zcytor17 truncated within the fibronectin domain isshown in SEQ ID NO:15 and the corresponding polypeptide sequence isshown in SEQ ID NO:18.

[0225] B. Isolation of a cDNA Coding for a Form of zcytor17 Truncated atthe End of the Cytokine-binding Domain

[0226] A 3′ RACE product for a truncated form of zcytor17 was generatedusing the HPVS cDNA library as a template and ZC27,895 (SEQ ID NO:14)and ZC6,346 (SEQ ID NO:11) as primers. This first-round 3′ RACE PCRreaction was run as follows: 30 cycles at 94° C. for 1 minute, 65° C.for 1 minute, 72° C. for 2 minutes, then 72° C. for 7 minutes; 4° C.soak. An aliquot of 3′ RACE PCR product was removed and analyzed on a1.0% agarose gel. Multiple bands were seen on the gel.

[0227] The remaining DNA was ethanol precipitated and diluted 1:40. Asecond-round nested 3′ RACE PCR reaction was run to amplify templatecDNA sequence. This PCR reaction used oligonucleotides ZC27,897 (SEQ IDNO:19) and ZC5,020 (SEQ ID NO:15), which were designed to anneal tosequence internal of ZC27,895 (SEQ ID NO:14) and ZC6,346 (SEQ ID NO:11).This nested PCR reaction was run as per the first-round 5′ RACEdisclosed in Example 1A, above. The resulting DNA products wereelectrophoresed on a 1.0% agarose gel and a prominent band atapproximately 1100 bp was seen. The DNA band was gel purified andsequenced. Sequence analysis revealed that the DNA product included partof the genomic AQ002781 DNA sequence (Genbank) and appeared to extendthe cDNA sequence for zcytor17 on the 3′ end to include a translationstop codon at the end of the cytokine-binding domain.

[0228] To confirm that the above sequence did indeed overlap with thegenomic AQ002781 DNA sequence, an additional PCR reaction was performed.A PCR product was generated using the HPVS cDNA library as a templateand oligonucleotides ZC28,481 (SEQ ID NO:10) and ZC28,521 (SEQ ID NO:20)as primers. The PCR reaction was run as follows: 30 cycles at 94° C. for1 minute, 65° C. for 1 minute, 72° C. for 2 minutes, then 72° C. for 7minutes; 4° C. soak. The resulting DNA products were electrophoresed ona 1.0% agarose gel and a prominent band at approximately 800 bp wasseen. The DNA band was gel purified and sequenced. Sequence analysesconfirmed that this was a truncated form of zcytor17. This couldrepresent an expressed soluble form of the receptor truncated near theend of the cytokine-binding domain. The polynucleotide sequence of thissoluble form of zcytor17 is shown in SEQ ID NO:21 and the correspondingpolypeptide sequence is shown in SEQ ID NO:22).

[0229] Another truncated 3′ RACE product was isolated using the protocoldescribed above for isolation of a cDNA variant truncated at thefibronectin domain (Example 2A). Sequencing of the isolated PCR productverified the sequence of the soluble form of zcytor17 as shown in SEQ IDNO:21.

Example 3

[0230] Tissue Distribution of Human zcytor17 in Tissue Panels UsingNorthern Blot and PCR

[0231] A. Human zcytor17 Tissue Distribution Using Northern Blot

[0232] Human Multiple Tissue Northern Blots (Human 12-lane MTN Blot Iand II, and Human Immune System MTN Blot II; Human Endocrine MTN, HumanFetal MTN Blot II, Human Multiple Tissue Array) (Clontech) as well as inhouse blots containing various tissues were probed to determine thetissue distribution of human zcytor17 expression. The in-house preparedblots included the following tissue and cell line mRNA: SK-Hep-1 cells,THP1 cells, Adrenal gland (Clontech); Kidney (Clontech), Liver (Clontechand Invitrogen); Spinal cord (Clontech), Testis (Clontech), Human CD4+T-cells, Human CD8+ T-cells, Human CD19+ T-cells, human mixed lymphocytereaction (MLR), THP1 cell line (ATCC No. TIB-202), U937 cell line,p388D1 mouse lymphoblast cell line (ATCC No. CCL-46) with or withoutstimulation by lonomycin; and WI-38 human embryonic lung cell line (ATCCNo. CRL-2221) with or without stimulation by Ionomycin.

[0233] An approximately 500 bp PCR derived probe was amplified using the5′ RACE (Example 1A) (SEQ ID NO:9) as template and oligonucleotidesZC28,575 (SEQ ID NO:23) and ZC27,899 (SEQ ID NO:24) as primers. The PCRamplification was carried out as follows: 30 cycles of 94° C. for 1minute, 65° C. for 1 minute, and 72° C. for 1 minute; followed by 1cycle at 72° C. for 7 minutes. The PCR product was visualized by agarosegel electrophoresis and the approximately 500 bp PCR product was gelpurified as described herein. The probe was radioactively labeled usingthe PRIME IT II™ Random Primer Labeling Kit (Stratagene) according tothe manufacturer's instructions. The probe was purified using a NUCTRAP™push column (Stratagene). EXPRESSHYB™ (Clontech) solution was used forthe prehybridization and as a hybridizing solution for the Northernblots. Prehybridization was carried out at 68° C. for 2 hours.Hybridization took place overnight at 68° C. with about 1.5×10⁶ cpm/mlof labeled probe. The blots were washed three times at room temperaturein 2×SSC, 0.05% SDS, followed by 1 wash for 10 minutes in 2×SSC, 0.1%SDS at 50° C. Several faint bands were seen after several days exposure.An approximately 9 kb transcript was seen in trachea, skeletal muscleand thymus; an approximately 2 kb transcript was seen in PBL, HPV, U937and THP-1 cells; and about a 1.2 kb transcript was seen in placenta,bone marrow and thyroid, and HPV and U937 cells. In all the tissueslisted above, the signal intensity was faint. There appeared to belittle expression in most normal tissues, suggesting that zcytor17expression may be dependent on activation of the cell or tissues inwhich it is expressed.

[0234] Northern analysis is also performed using Human Cancer Cell LineMTN™ (Clontech). PCR and probing conditions are as described above. Astrong signal in a cancer line suggests that zcytor17 expression may beexpressed in activated cells and/or may indicate a cancerous diseasestate. Moreover, using methods known in the art, Northern blots or PCRanalysis of activated lymphocyte cells can also show whether zcytor17 isexpressed in activated immune cells.

[0235] B. Tissue Distribution in Tissue Panels Using PCR

[0236] A panel of cDNAs from human tissues was screened for zcytor17expression using PCR. The panel was made in-house and contained 94marathon cDNA and cDNA samples from various normal and cancerous humantissues and cell lines is shown in Table 5, below. The cDNAs came fromin-house libraries or marathon cDNAs from in-house RNA preps, ClontechRNA, or Invitrogen RNA. The marathon cDNAs were made using themarathon-Ready™ kit (Clontech, Palo Alto, Calif.) and QC tested withclathrin primers ZC21195 (SEQ ID NO:49) and ZC21196 (SEQ ID NO:50) andthen diluted based on the intensity of the clathrin band. To assurequality of the panel samples, three tests for quality control (QC) wererun: (1) To assess the RNA quality used for the libraries, the in-housecDNAs were tested for average insert size by PCR with vector oligos thatwere specific for the vector sequences for an individual cDNA library;(2) Standardization of the concentration of the cDNA in panel sampleswas achieved using standard PCR methods to amplify full length alphatubulin or G3PDH cDNA using a 5′ vector oligo ZC14,063 (SEQ ID NO:25)and 3′ alpha tubulin specific oligo primer ZC17,574 (SEQ ID NO:26) or 3′G3PDH specific oligo primer ZC17,600 (SEQ ID NO:27); and (3) a samplewas sent to sequencing to check for possible ribosomal or mitochondrialDNA contamination. The panel was set up in a 96-well format thatincluded a human genomic DNA (Clontech, Palo Alto, Calif.) positivecontrol sample. Each well contained approximately 0.2-100 pg/μl of cDNA.The PCR reactions were set up using oligos ZC26,358 (SEQ ID NO:28) andZC26,359 (SEQ ID NO:29), TaKaRa Ex Taq™ (TAKARA Shuzo Co LTD,Biomedicals Group, Japan), and Rediload dye (Research Genetics, Inc.,Huntsville, Ala.). The amplification was carried out as follows: 1 cycleat 94° C. for 2 minutes, 35 cycles of 94° C. for 30 seconds, 66.3° C.for 30 seconds and 72° C. for 30 seconds, followed by 1 cycle at 72° C.for 5 minutes. About 10 μl of the PCR reaction product was subjected tostandard Agarose gel electrophoresis using a 4% agarose gel. The correctpredicted DNA fragment size was observed in lymph node, prostate,thyroid, HPV (prostate epithelia), HPVS (prostate epithelia, selected),lung tumor, uterus tumor reactions, along with the genomic DNA reaction.One of the primers can anneal to genomic or to the zcytor17 short-formsoluble receptor (SEQ ID NO:21), suggesting that the expression patternseen may be that of this alternative form of zcytor17.

[0237] The DNA fragment for prostate tissue (2 samples), HPV (prostateepithelia), HPVS (prostate epithelia, selected), and genomic wereexcised and purified using a Gel Extraction Kit (Qiagen, Chatsworth,Calif.) according to manufacturer's instructions. Fragments wereconfirmed by sequencing to show that they were indeed zcytor17. TABLE 5Tissue/Cell line #samples Tissue/Cell line #samples Adrenal gland 1 Bonemarrow 3 Bladder 1 Fetal brain 3 Bone Marrow 1 Islet 2 Brain 1 Prostate3 Cervix 1 RPMI #1788 2 (ATCC # CCL-156) Colon 1 Testis 4 Fetal brain 1Thyroid 2 Fetal heart 1 WI38 (ATCC # CCL-75 2 Fetal kidney 1 ARIP (ATCC# CRL-1674 - rat) 1 Fetal liver 1 HaCat - human keratinocytes 1 Fetallung 1 HPV (ATCC # CRL-2221) 1 Fetal muscle 1 Adrenal gland 1 Fetal skin1 Prostate SM 2 Heart 2 CD3+ selected PBMC's 1 Ionomycin + PMAstimulated K562 (ATCC 1 HPVS (ATCC # CRL-2221) - 1 # CCL-243) selectedKidney 1 Heart 1 Liver 1 Pituitary 1 Lung 1 Placenta 2 Lymph node 1Salivary gland 1 Melanoma 1 HL60 (ATCC # CCL-240) 3 Pancreas 1 Platelet1 Pituitary 1 HBL-100 1 Placenta 1 Renal mesangial 1 Prostate 1 T-cell 1Rectum 1 Neutrophil 1 Salivary Gland 1 MPC 1 Skeletal muscle 1 Hut-102(ATCC # TIB-162) 1 Small intestine 1 Endothelial 1 Spinal cord 1 HepG2(ATCC # HB-8065) 1 Spleen 1 Fibroblast 1 Stomach 1 E. Histo 1 Testis 2Thymus 1 Thyroid 1 Trachea 1 Uterus 1 Esophagus 1 tumor Gastric tumor 1Kidney tumor 1 Liver tumor 1 Lung tumor 1 Ovarian tumor 1 Rectal tumor 1Uterus tumor 1

[0238] B. Expression Analysis of zcytoR17 by PCR and Northern

[0239] Annotation of the cell types and growth conditions that affectexpression of the receptor is a useful means of elucidating its functionand predicting a source of ligand. To that end we surveyed a widevariety of tissue and cell types by PCR. The thermostable polymeraseAdvantage II™ (Clontech, La Jolla, Calif.) was used with theoligonucleotide primers ZC29,180 (SEQ ID NO:73) and ZC29,179 (SEQ IDNO:74) and 1-10 ng of the various cDNA templates listed below for 30amplification cycles of (94° C., 30 sec.; 66° C., 20 sec.; 68° C., 1min. 30 sec.). Following this, 20% of each reaction was run out on 0.8%agarose, TAE/ethidium bromide gels and visualized with UV light. Sampleswere then scored on the basis of band intensity. See Table 6 below.TABLE 6 Cells and Conditions Score 0-5 Hel stimulated with PMA 0 U937 3MCF-7 0 HuH7 1 Human follicle 0 HT-29 0 HEPG2 0 HepG2 stimulated withIL6 0 Human dermal endothelial 0 Human venous endothelial 0 Human CD4+ 0BEWO 0 Human CD19+ 1 Human PBMC stimulated with PHA, PMA, Ionomycin, 0IL2, IL4, TNFα 24 hours Human PBMC stimulated with LPS, PWM, IFNγ, 0TNFα, 24 hours Human PBMC all of the above conditions for 48 hours 4HUVEC p.2 4 RPMI1788 0 TF1 0 Monkey spleen T cells stimulated with PMA,Ionomycin 0 Human prostate epithelia HPV transformed 5 Human tonsils,inflamed 0 HACAT 0 Human chondrocyte 1 Human synoviacyte 1 THP1 5 REH 0

[0240] Of the strong positive PCR signals, two were from the humanmonocyte cell lines U937 and THP1.

[0241] These two cell lines along with a prostate epithelia line wereselected for further analysis by Northern blot. Previous attempts atvisualizing a transcript by northern analysis using mRNA from varioustissues yielded weak and diffuse signals in the suprisingly large sizerange of 7-10 kb making this data difficult to interpret. A denaturingformaldehyde/MOPS/0.8% agarose gel was prepared (RNA Methodologies,Farrell, RE Academic Press) and 2 μg of polyA+ mRNA was run for eachsample along side an RNA ladder (Life Technologies, Bethesda, Md.). Thegel was then transferred to Hybond nylon (Amersham, Buckinghamshire,UK), UV crosslinked, and hybridized in ExpressHyb solution (Clontech,LaJolla, Calif.) at 68° C. overnight using a probe to human zcytoR17generated by PCR with the oligos ZC28,575 (SEQ ID NO:23), and ZC27,899(SEQ ID NO:24) and labeled with a Megaprime ³²P kit (Amersham). Thenorthern blot was subsequently washed with 0.2×SSC+0.1% SDS at 65 C. for15 minutes and exposed to film for 7 days with intensifying screens. Aprominent 8 kb band was seen in both the prostate epithelia and U937lanes while a fainter band was present in the THP1 lane.

[0242] To optimize the cDNA used as a hybridization probe, fourdifferent regions of the full-length human zcytoR17 sequence wereamplified by PCR, labeled and hybridized as described above to southernblots containing genomic and amplified cDNA library DNA. The fourprobes, herein designated probes A-D, were amplified using the followingprimer pairs: (A) ZC28,575 (SEQ ID NO:23), ZC27,899 (SEQ ID NO:24); (B)ZC27,895 (SEQ ID NO:64), ZC28,917 (SEQ ID NO:73); (C) ZC28,916 (SEQ IDNO:75), ZC28,918 (SEQ ID NO:76); and (D) ZC28,916 (SEQ ID NO:75),ZC29,122 (SEQ ID NO:65). Human genomic DNA along with amplified cDNAlibraries demonstrated to contain zcytor17 by PCR were digested withEcoR1 and Xho1 to liberate inserts and run out on duplicate TAE/0.8%agarose gels, denatured with 0.5M NaOH, 1.5 M NaCl, blotted to Hybond,UV crosslinked and each hybridized with a distinct probe. Probe B wasfound to have the least nonspecific binding and strongest signal. Thus,Probe B was used for all subsequent hybridizations.

[0243] Given that the THP1 cells are an excellent model of circulatingmonocytes and expressed zcytor17 at low levels we treated them with avariety of compounds in an effort to increase expression of zcytoR17.The cells were grown to a density of 2e5/ml, washed and resuspended invarious stimulating media, grown for four or thirty hours, and harvestedfor RNA preparations. Each media was supplemented with one of thefollowing drugs or pairs of cytokines: LPS 2 ug/ml (Sigma Chemicals,StLouis Mo.), hTNFα 2 ng/ml (R&D Systems,Minneapolis,Minn.), hGMCSF 2ng/ml (R&D Systems,Minneapolis,Minn.), hIFNγ 50 ng/ml (R&DSystems,Minneapolis,Minn.), hMCSF 1 ng/ml (R&DSystems,Minneapolis,Minn.), hIL6 1 ng/ml (R&DSystems,Minneapolis,Minn.), hIL1β 2 ng/ml (R&DSystems,Minneapolis,Minn.), hIFNγ 50 ng/ml+hIL4 0.5 ng/ml (R&DSystems,Minneapolis,Minn.), hIFNγ 50 ng/ml+hIL10 1 ng/ml (R&DSystems,Minneapolis,Minn.), PMA 10 ng/ml (Calbiochem, SanDiego,Calif.)and an untreated control. At the end of the culture period Total RNA wasprepared using an RNAeasy Midi-kit (Qiagen, Valencia, Calif.). Poly A+RNA was selected from the total RNA using an MPG kit (CPG, Lincoln Park,N.J.). 2 ug of polyA+ RNA from each condition was run onformaldehyde/MOPS/agarose gels, transferred to nylon and UV crosslinkedas described above. These northern blots were then hybridized, as above,to probe B at 68° C. overnight, washed at high stringency with 0.2×SSC,0.1% SDS at 65 C, exposed to film overnight then exposed to phosphorscreens for signal quantitation (see FIG. 2). A dominant 8 kb mRNA aswell a relatively weaker 2.8 kb band were seen in all lanes. A 20-foldincrease in zcytor17 mRNA was seen in RNA from cells treated with hIFNγfor 30 hours, this effect was slightly muted with simultaneous treatmentwith IL4. Minor 3 fold increases in mRNA were seen in RNA from cellstreated with LPS, TNFα and GM-CSF while MCSF, IL6, and IL1β had noeffect on zcytor17 mRNA levels. Taken together this data suggests a rolefor the zcytor17 receptor and its ligand in monocyte macrophage biologyand by extension any number of disease processes in which these cellparticipate.

Example 4

[0244] PCR-Based Chromosomal Mapping of the zcytor17 Gene

[0245] Zcytor17 was mapped to chromosome 5 using the commerciallyavailable “GeneBridge 4 Radiation Hybrid (RH) Mapping Panel” (ResearchGenetics, Inc., Huntsville, Ala.). The GeneBridge 4 RH panel containsDNA from each of 93 radiation hybrid clones, plus two control DNAs (theHFL donor and the A23 recipient). A publicly available WWW server(http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) allows mappingrelative to the Whitehead Institute/MIT Center for Genome Research'sradiation hybrid map of the human genome (the “WICGR” radiation hybridmap) which was constructed with the GeneBridge 4 RH panel.

[0246] For the mapping of Zcytor17 with the GeneBridge 4 RH panel, 20 μlreactions were set up in a 96-well microtiter plate compatible for PCR(Stratagene, La Jolla, Calif.) and used in a “RoboCycler Gradient 96”thermal cycler (Stratagene). Each of the 95 PCR reactions consisted of 2μl 10×KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., PaloAlto, Calif.), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City,Calif.), 1 μl sense primer, ZC27,895 (SEQ ID NO:14), 1 μl antisenseprimer, ZC27,899 (SEQ ID NO:24), 2 μl “RediLoad” (Research Genetics,Inc., Huntsville, Ala.), 0.4 μl 5×Advantage KlenTaq Polymerase Mix(Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybridclone or control and distilled water for a total volume of 20 μl. Thereactions were overlaid with an equal amount of mineral oil and sealed.The PCR cycler conditions were as follows: an initial 1 cycle 5 minutedenaturation at 94° C., 35 cycles of a 45 seconds denaturation at 94°C., 45 seconds annealing at 54° C. and 1 minute AND 15 seconds extensionat 72° C., followed by a final 1 cycle extension of 7 minutes at 72° C.The reactions were separated by electrophoresis on a 2% agarose gel (EMScience, Gibbstown, N.J.) and visualized by staining with ethidiumbromide.

[0247] The results showed that Zcytor17 maps 6.72 cR_(—)3000 distal fromthe framework marker AFM183YB8 on the chromosome 5 WICGR radiationhybrid map. The use of surrounding genes/markers positions Zcytor17 inthe 5q11 chromosomal region.

Example 5

[0248] Construction of MPL-zcytor17 Polypeptide Chimera: MPLExtracellular and TM Domain Fused to the zcytor17 IntracellularSignaling Domain

[0249] The 5′ extracellular domain of the murine MPL receptor wasisolated from a plasmid containing the murine MPL receptor (PHZ1/MPLplasmid) by digestion with EcoRI and BamHI generating a 1164 bpfragment. The digestion was run on a 1% agarose gel and the fragment wasisolated using the Qiaquick gel extraction kit (Qiagen) as permanufacturer's instructions. The rest of the MPL extracellular domainand transmembrane domain were generated using PCR with primers ZC6,673(SEQ ID NO:58) and ZC29,082 (SEQ ID NO:59). The reaction conditions wereas follows: 15 cycles at 94° C. for 1 min., 55° C. for 1 min., 72° C.for 2 min.; followed by 72° C. for 7 min.; then a 4° C. soak. The PCRproduct was run on a 1% agarose gel and the approximately 400 bp MPLreceptor fragment was isolated using Qiaquick™ gel extraction kit(Qiagen) as per manufacturer's instructions.

[0250] The intracellular domain of human zcytor17 was isolated from aplasmid containing zcytor17 receptor cDNA (#23/pCAP) using PCR withprimers ZC29,083 (SEQ ID NO:60) and ZC29,145 (SEQ ID NO:61). Thepolynucleotide sequence corresponds to the zcytor17 receptor codingsequence is shown in SEQ ID NO:54. The reaction conditions were as perabove. The PCR product was run on a 1% agarose gel and the approximately320 bp zcytor17 fragment isolated using Qiaquick gel extraction kit asper manufacturer's instructions.

[0251] Each of the isolated PCR fragments described above were mixed ata 1:1 volumetric ratio and used in a PCR reaction using ZC6673 (SEQ IDNO:58) and ZC29145 (SEQ ID NO:61) to create all but the 5′ MPL portionof the MPL-zcytor17 chimera. The reaction conditions were as follows: 15cycles at 94° C. for 1 min., 55° C. for 1 min., 72° C. for 2 min.;followed by 72° C. for 7 min.; then a 4° C. soak. The entire PCR productwas run on a 1% agarose gel and the approximately 700 bp MPL-zcytor17chimera fragment isolated using Qiaquick gel extraction kit (Qiagen) asper manufacturer's instructions. The MPL-zcytor17 chimera fragment wasdigested with BamHI (BRL) and XbaI (Boerhinger Mannheim) as permanufacturer's instructions. The entire digest was run on a 1% agarosegel and the cleaved MPL-zcytor17 chimera isolated using Qiaquick™ gelextraction kit (Qiagen) as per manufacturer's instructions. Theresultant cleaved MPL-zvytor17 chimera plus 5′ MPL EcoRI/BamHI fragmentdescribed above were inserted into an expression vector to generate thefull MPL-zcytor17 chimeric receptor as described below.

[0252] Recipient expression vector pZP-7 was digested with EcoRI (BRL)and Xba1 (BRL) as per manufacturer's instructions, and gel purified asdescribed above. This vector fragment was combined with the EcoRI andXbaI cleaved MPL-zcytor17 PCR chimera isolated above and the EcoRI andBamHI 5′ MPL fragment isolated above in a ligation reaction. Theligation was run using T4 Ligase (Epicentre Technologies), at roomtemperature for 1 hour as per manufacturer's instructions. A sample ofthe ligation was electroporated into DH10B ElectroMAX™ electrocompetentE. coli cells (25 μF, 200Ω, 1.8V). Transformants were plated onLB+Ampicillin plates and single colonies screened by miniprep (Qiagen)and digestion with EcoRI to check for the MPL-zcytor17 chimera. EcoRIdigestion of correct clones yield about a 2 kb fragment. Confirmation ofthe MPL-zcytor17 chimera sequence was made by sequence analyses. Theinsert was approximately 3.1 kb, and was full-length.

Example 6

[0253] MPL-zcytor17 Chimera Based Proliferation in BAF3 Assay UsingAlamar Blue

[0254] A. Construction of BaF3 Cells Expressing MPL-zcytor17 Chimera

[0255] BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell linederived from murine bone marrow (Palacios and Steinmetz, Cell 41:727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135,1986), was maintained in complete media (RPMI medium (JRH BioscienceInc., Lenexa, Kans.) supplemented with 10% heat-inactivated fetal calfserum, 1 ng/ml murine IL-3 (mIL-3) (R & D, Minneapolis, Minn.), 2 mML-glutaMax-1™ (Gibco BRL), 1 mM Sodium Pyruvate (Gibco BRL), and PSNantibiotics (GIBCO BRL)). Prior to electroporation, pZP-7/MPL-zcytor17plasmid DNA (Example 5) was prepared and purified using a Qiagen MaxiPrep kit (Qiagen) as per manufacturer's instructions. BaF3 cells forelectroporation were washed twice in RPMI media and then resuspended inRPMI media at a cell density of 10⁷ cells/ml. One ml of resuspended BaF3cells was mixed with 30 μg of the pZP-7/MPL-zcytor17 plasmid DNA andtransferred to separate disposable electroporation chambers (GIBCO BRL).At room temperature cells were given 5×0.1 msec shocks at 800 voltsfollowed by 5×2 ms shocks at 600 volts delivered by an electroporationapparatus (Cyto-Pulse). The electroporated cells were transferred to 50ml of complete media and placed in an incubator for 15-24 hours (37° C.,5% CO₂). Then Geneticin™ (Gibco) selection (1 mg/ml G418) was added tothe cells in a T-162 flask to isolate the G418-resistant pool. Pools ofthe transfected BaF3 cells, hereinafter called BaF3/MPL-zcytor17 cells,were assayed for signaling capability as described below.

[0256] B. Testing the Signaling Capability of the BaF3/MPL-zcytor17Cells Using an Alamar Blue Proliferation Assay

[0257] BaF3/MPL-zcytor17 cells were spun down and washed in the completemedia, described above, but without mIL-3 (hereinafter referred to as“mIL-3 free media”). The cells were spun and washed 3 times to ensurethe removal of the mIL-3. Cells were then counted in a hemacytometer.Cells were plated in a 96-well format at 5000 cells per well in a volumeof 100 μl per well using the mIL-3 free media.

[0258] Proliferation of the BaF3/MPL-zcytor17 cells was assessed usingmurine thrombopoietin (mTPO) diluted with mIL-3 free media to 200 ng/ml,100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.1 ng/ml, 1.5ng/ml concentrations. 100 μl of the diluted mTPO was added to theBaF3/MPL-zcytor17 cells. The total assay volume is 200 μl. Negativecontrols were run in parallel using mIL-3 free media only, without theaddition of mTPO. The assay plates were incubated at 37° C., 5% CO₂ for3 days at which time Alamar Blue (Accumed, Chicago, Ill.) was added at20 μl/well. Alamar Blue gives a fluorometric readout based on themetabolic activity of cells, and is thus a direct measurement of cellproliferation in comparison to a negative control. Plates were againincubated at 37° C., 5% CO₂ for 24 hours. Plates were read on the Fmax™plate reader (Molecular Devices Sunnyvale, Calif.) using the SoftMax™Pro program, at wavelengths 544 (Excitation) and 590 (Emission).

[0259] Results confirmed the signaling capability of the intracellularportion of the zcytor17 receptor, as the thrombopoietin inducedproliferation at approximately 9-13 fold over background at mTPOconcentrations of 50 ng/ml and greater.

Example 7

[0260] Construction of Zcytor17-mpl Polypeptide Chimera: Zcytor17Extracellular Domain Fused to the Mpl Intracellular Signaling Domain andTM Domain

[0261] The extracellular domains of the zcytor17 receptor are isolatedfrom a plasmid containing the zcytor17 receptor using PCR with primersdesigned to amplify the extracellular domain or portion thereof ofzcytor17 shown in SEQ ID NO:1, SEQ ID NO:45, SEQ ID NO:17 or SEQ IDNO:21 or corresponding region of SEQ ID NO:53 or SEQ ID NO:56. Preferredreaction conditions are as follows: 95° C. for 1 min.; 35 cycles at 95°C. for 1 min., 45° C. for 1 min., 72° C. for 2 min.; followed by 72° C.at 10 min.; then a 10° C. soak. The PCR product is run on a 1% lowmelting point agarose (Boerhinger Mannheim, Indianapolis, Ind.) and thezcytor17 receptor fragment isolated using Qiaquick™ gel extraction kit(Qiagen) as per manufacturer's instructions.

[0262] The intracellular and transmembrane domains of MPL are isolatedfrom a plasmid containing MPL receptor cDNA (PHZ1/MPL plasmid) (Example5) using PCR with primers spanning the 3′ end of the zcytor17extracellular domain and the 5′ end of the MPL intracellular andtransmembrane domains and ZC17,206 (SEQ ID NO:33). Preferred reactionconditions are run as per above. The PCR product is run on a 1% lowmelting point agarose (Boerhinger Mannheim) and the approximately 450 bpMPL fragment isolated using Qiaquick gel extraction kit (Qiagen) as permanufacturer's instructions.

[0263] Each of the isolated fragments described above are mixed at a 1:1volumetric ratio and used in a PCR reaction using the 5′ primer used toamplify the extracellular domain of zcytor17 and ZC17,206 (SEQ ID NO:33)to create a Zcytor17-mpl chimera. Preferred reaction conditions are asfollows: 95° C. for 1 min.; 35 cycles at 95° C. for 1 min., 55° C. for 1min., 72° C. for 2 min.; followed by 72° C. at 10 min.; then a 10° C.soak. The entire PCR product is run on a 1% low melting point agarose(Boehringer Mannheim) and an approximately 1.2 kb Zcytor17-mpl chimerafragment isolated using Qiaquick gel extraction kit (Qiagen) as permanufacturer's instructions. The Zcytor17-mpl chimera fragment isdigested with, e.g., EcoRI (BRL) and XbaI (Boerhinger Mannheim) as permanufacturer's instructions. The entire digest is run on a 1% lowmelting point agarose (Boehringer Mannheim) and the cleaved Zcytor17-mplchimera isolated using Qiaquick™ gel extraction kit (Qiagen) as permanufacturer's instructions. The resultant cleaved Zcytor17-mpl chimerais inserted into an expression vector as described below.

[0264] Recipient expression vector pZP-5Z is digested with EcoRI (BRL)and HindIII (BRL) as per manufacturer's instructions, and gel purifiedas described above. This vector fragment is combined with the EcoRI andXbaI cleaved Zcytor17-mpl chimera isolated above and a XbaI/HindIIIlinker fragment in a ligation reaction. The ligation is run using T4Ligase (BRL), at 15° C. overnight. A sample of the ligation iselectroporated in to DH10B ElectroMAX™ electrocompetent E. coli cells(25 μF, 200Ω, 2.3V). Transformants are plated on LB+Ampicillin platesand single colonies screened by PCR to check for the Zcytor17-mplchimera using a zcytor17 extracellular domain primer and and ZC 17,206(SEQ ID NO:25) using the PCR conditions as described above. Confirmationof the Zcytor17-mpl chimera sequence is made by sequence analyses.

Example 8

[0265] Construction of Expression Vector Expressing Full-lengthzcytor17: pZp7pX/zcytor17

[0266] A. Cloning of Full Length zcytor17 cDNA for Expression

[0267] To obtain a full-length cDNA, 5′ and 3′ PCR products wereisolated and joined using an internal PstI site. The PCR primers weredesigned using the nucleotide sequence SEQ ID NO:53 and include BamHIand Xho I restriction sites for cloning purposes.

[0268] A 5′ PCR product was generated using a WI-38 cDNA library as atemplate and oligonucleotides ZC 29,359 (SEQ ID NO:62) and ZC 27,899(SEQ ID NO:63) as primers. WI-38 is an in-house cDNA library generatedfrom a human embryonic lung cell line (ATCC CRL-2221). This 5′ PCRreaction was run as follows: 30 cycles at 94° C. for 1 minute, 65° C.for 1 minute, 72° C. for 2 minutes, then 72° C. for 7 minutes; 10° C.soak. The PCR reaction used approximately 3 ug of plasmid prepared fromthe cDNA library, 20 pmoles of each oligonucleotide, and five units ofPWO DNA polymerase (Roche). About 90% of the 5′ PCR product was ethanolprecipitated, digested with BamHI and PstI and gel purified on a 1.0%agarose gel. The approximately 600 bp band was excised and used forligation to the cloning vector pUC18 digested with BamHI and PstI. Theresulting transformants were sequenced to confirm the zcytor17 cDNAsequence. For one of these transformants, plasmid DNA was prepared anddigested with BamHI and PstI. The resulting approximately 600 bp bandwas gel purified and used for a ligation below to form a full-lengthcDNA.

[0269] A 3′ PCR product was generated using a human testes in-house cDNAlibrary as a template and oligonucleotides ZC 27,895 (SEQ ID NO:64) andZC 29,122 (SEQ ID NO:65) as primers. This 3′ PCR reaction was run asfollows: 30 cycles at 94° C. for 45 seconds, 65° C. for 45 seconds, 72°C. for 2 minutes, then 72° C. for 7 minutes; 10° C. soak. The entire 3′PCR reaction was gel purified on a 1.0% agarose gel and the major 1500bp band excised. This band was cloned into the PCR Blunt II TOPO vectorusing the Zeroblunt TOPO kit (Invitrogen). The resulting transformantswere sequenced to confirm the zcytor17 cDNA sequence. For one of thesetransformants, plasmid DNA was prepared and digested with PstI and XhoI.The resulting approximately 1500 bp band was gel purified. A three-partligation was performed with the 5′ BamHI to Pst I fragment above, the 3′PstI to XhoI fragment, and the expression vector pZp7pX digested withBaMHI and XhoI. This generated a pZp7pX plasmid containing a full-lengthcDNA for zcytor17 (SEQ ID NO:53), designated pZp7p/zcytor17. The fulllength zcytor17 cDNA in pZp7p/zcytor17 has a silent mutations thatchange the T to G at position 1888 of SEQ ID NO:53 (encoding a Glyresidue at residue 464 of SEQ ID NO:54). As this mutation is silent, thezcytor17 cDNA in pZp7p/zcytor17 encodes the polypeptide as shown in SEQID NO:54. Plasmid pZp7pX is a mammalian expression vector containing anexpression cassette having the CMV promoter, intron A, multiplerestriction sites for insertion of coding sequences, and a human growthhormone terminator. The plasmid also has an E. coli origin ofreplication, a mammalian selectable marker expression unit having anSV40 promoter, enhancer and origin of replication, a puromycinresistance gene and the SV40 terminator.

Example 9

[0270] Construction of Cells to Assess zcytor17 Based Proliferation inBAF3 Assay Using Alamar Blue

[0271] A. Construction of BaF3 Cells Expressing Zcytor17-MPL receptor

[0272] BaF3 cells expressing the Zcytor17-MPL receptor are constructedas per Example 6A, using 30 μg of the zcytor17 expression vector,described in Example 7. The BaF3 cells expressing the pZP-5Z/zcytor17receptor plasmid are designated as BaF3/Zcytor17-mpl. These cells areused to screen for a zcytor17 activity as described below in Examples 10and 18.

[0273] B. Construction of BaF3 Cells Expressing zcytor17 Receptor

[0274] BaF3 cells expressing the full-length zcytor17 receptor areconstructed as per Example 6A, using 30 μg of the zcytor17 expressionvector, described in Example 8. The BaF3 cells expressing thepZp7p/zcytor17 receptor plasmid are designated as BaF3/zcytor17. Thesecells are used to screen for a zcytor17 activity as described below inExamples 10 and 18.

Example 10

[0275] Screening for zcytor17 Activity Using BaF3/zcytor17-MPL Cells andBaf3/zcytor17 Cells Using an Alamar Blue Proliferation Assay

[0276] Baf3/zcytor17-mpl chimera cells and Baf3/zcytor17 cells (Example9) are spun down and washed independently in mIL-3 free media (Example6). The cells are spun and washed 3 times to ensure the removal of themIL-3. Cells are then counted in a hemacytometer. Cells are plated in a96-well format at 5000 cells per well in a volume of 100 μl per wellusing the mIL-3 free media.

[0277] To try and identify a source for the zcytor17 ligand,approximately 124 conditioned media and samples from a variety of celllines and tissues are screened. 100 μl of each conditioned media sampleis added to the BaF3/MPL-zcytor17 chimera cells as well as theBaf3/zcytor17 cells. The total assay volume is 200 μl. All knowncytokines are also screened at a concentration of about 100 pg/ml-250ng/ml on both cell lines. Negative controls are run in parallel usingmIL-3 free media only. Mouse IL-3 at a concentration of 250 pg/ml isused as a positive control. The assay plates are incubated at 37° C., 5%CO₂ for 3 days at which time Alamar Blue (Accumed, Chicago, Ill.) isadded at 20 μl/well. Alamar Blue gives a fluorometric readout based onnumber of live cells, and is thus a direct measurement of cellproliferation in comparison to a negative control. Plates are againincubated at 37° C., 5% CO₂ for 24 hours. Plates are read on the Fmax™plate reader (Molecular Devices Sunnyvale, Calif.) using the SoftMax™Pro program, at wavelengths 544 (Excitation) and 590 (Emission).

[0278] Results that show proliferation of on either theBaf3/zcytor17-mpl chimera cell line or the Baf3/zcytor17 cell line inresponse to conditioned media samples or the known ligands identify asource for the ligand, and suggest that the zcytor17 receptor may signalas a homodimer, or heterodimerize or multimerize with a receptor presentin the BaF3 cells. If no signal is present, the actualreceptor-signaling complex may heterodimerize or multimerize withanother receptor subunit not present in BaF3 cells. See example 18 andExample 19 below.

Example 11

[0279] Construction of Mammalian Expression Vectors that Expresszcytor17 Soluble Receptors: zcytor17CEE, zcytor17CFLG, zcytor17CHIS andzcytor17-Fc4

[0280] A. Construction of zcytor17 Mammalian Expression VectorContaining zcytor17CEE, zcytor17CFLG and zcytor17CHIS

[0281] An expression vector was prepared for the expression of thesoluble, extracellular domain of the zcytor17 polypeptide,pZp9zcytor17CEE, where the construct is designed to express a zcytor17polypeptide comprised of the predicted initiating methionine andtruncated adjacent to the predicted transmembrane domain, and with aC-terminal GLU-GLU tag (SEQ ID NO:34).

[0282] An approximately 1500 bp PCR product was generated using ZC29,451(SEQ ID NO:66) and ZC29,124 (SEQ ID NO:67) as PCR primers to add EcoRIand BamHI restriction sites. A human HPVS in-house cDNA library was usedas a template and PCR amplification was performed as follows: 30 cyclesat 94° C. for 1 minute, 65 °C. for 1 minute, 72° C. for 1.5 minutes,then 72° C. for 7 minutes; 10° C. soak. The PCR reaction was ethanolprecipitated and digested with EcoRI and BamHI restriction enzymes. Thedigested PCR product was gel purified on a 1.0% agarose gel and theapproximately 1500 bp band excised. This band was then re-amplifiedusing identical primers with the following cycling: 30 cycles at 94° C.for 1 minute, 65° C. for 1 minute, 72° C. for 3 minutes, then 72° C. for7 minutes; 10° C. soak. The PCR reaction was ethanol precipitated anddigested with EcoRI and BamHI restriction enzymes. The digested PCRproduct was gel purified on a 1.0% agarose gel and the approximately1500 bp band excised. The excised DNA was subcloned into plasmid CEEpZp9that had been cut with EcoRI and BamHI, to generate plasmid with aGLU-GLU C-terminally tagged soluble receptor for zcytor17,zyctor17CEEpZp9. The extracellular domain in the zyctor17CEE cDNA inzyctor17CEEpZp9 has a silent mutation that changes the T to C atposition 1705 of SEQ ID NO:53 (encoding a Pro residue at residue 403 ofSEQ ID NO:54). As this mutation is silent, the zcytor17 cDNA inzyctor17CEEpZp9 encodes the polypeptide as shown in SEQ ID NO:54.Moreover, because of the construct used, a Gly-Ser residue pair isinserted C-terminal to the end of the soluble, extracellular domain ofzcytor17 and prior to the C-terminal Glu-Glu Tag (SEQ ID NO:34). Assuch, the tag at the C-terminus of the zcytor17 extracellular domain,was a modified Glu-Glu tag as shown in (SEQ ID NO:91). Plasmid CEEpZp9is a mammalian expression vector containing an expression cassettehaving the mouse metallothionein-1 promoter, multiple restriction sitesfor insertion of coding sequences, and a human growth hormoneterminator. The plasmid also has an E. coli origin of replication, amammalian selectable marker expression unit having an SV40 promoter,enhancer and origin of replication, a DHFR gene and the SV40 terminator.Using standard molecular biological techniques zyctor17CEEpZp9 waselectroporated into DH10B competent cells (GIBCO BRL, Gaithersburg, Md.)according to manufacturer's direction and plated onto LB platescontaining 100 μg/ml ampicillin, and incubated overnight. Colonies werescreened by restriction analysis, or PCR from DNA prepared fromindividual colonies. The insert sequence of positive clones was verifiedby sequence analysis. A large scale plasmid preparation was done using aQIAGEN® Maxi prep kit (Qiagen) according to manufacturer's instructions.

[0283] The same process is used to prepare the zcytor17 solublereceptors with a C-terminal his tag, composed of 6 His residues in arow; and a C-terminal FLAG® tag (SEQ ID NO:35), zcytor17CFLAG. Toconstruct these constructs, the aforementioned vector has either the HISor the FLAG® tag in place of the glu-glu tag (SEQ ID NO:34).

[0284] B. Mammalian Expression Construction of Soluble Human zcytor17receptor: zcytor17-Fc4

[0285] An expression vector, pEZE-2 hzcytor17/Fc4, was prepared toexpress a C-terminally Fc4 tagged soluble version of hzcytor17 (humanzcytor17-Fc4) in PF CHO cells. PF CHO cells are an in house CHO cellline adapted for growth in protein-free medium (ExCell 325 PF medium;JRH Biosciences). The in house CHO cell line was originally derived fromCHO DG44 cells (G. Urlaub, J. Mitchell, E. Kas, L. A. Chasin, V. L.Funanage, T. T. Myoda and J. L. Hamlin, “The Effect Of Gamma Rays at theDihydrofolate Reductase Locus: Deletions and Inversions,” Somatic Celland Molec. Genet., 12: 555-566 (1986). A fragment of zcytor17 cDNA thatincludes the polynucleotide sequence from extracellular domain of thezcytor17 receptor was fused in frame to the Fc4 polynucleotide sequence(SEQ ID NO:36) to generate a zcytor17-Fc4 fusion (SEQ ID NO:68 and SEQID NO:69). The pEZE-2 vector is a mammalian expression vector thatcontains the Fc4 polynucleotide sequence and a cloning site that allowsrapid construction of C-terminal Fc4 fusions using standard molecularbiology techniques.

[0286] A 1566 base pair fragment was generated by PCR, containing theextracellular domain of human zcytor17 and the first two amino acids ofFc4 (Glu and Pro) with FseI and BglII sites coded on the 5′ and 3′ ends,respectively. This PCR fragment was generated using primers ZC29,157(SEQ ID NO:70) and ZC29,150 (SEQ ID NO:71) by amplification from aplasmid containing the extracellular domain of human zcytor17(pZp9zcytor17CEE) (Example 11A). The PCR reaction conditions were asfollows: 25 cycles of 94° C. for 1 minute, 60° C. for 1 minute, and 72°C. for 2 minutes; 1 cycle at 72° C. for 10 minutes; followed by a 4° C.soak. The fragment was digested with FseI and BglII restrictionendonucleases and subsequently purified by 1% gel electrophoresis andband purification using QiaQuick gel extraction kit (Qiagen). Theresulting purified DNA was ligated for 5 hours at room temperature intoa pEZE-2 vector previously digested with FseI and BglII containing Fc43′ of the FseI and BglII sites.

[0287] Two μl of the ligation mix was electroporated in 37 μl DH10Belectrocompetent E. coli (Gibco) according to the manufacturer'sdirections. The transformed cells were diluted in 400 μl of LB media andplated onto LB plates containing 100 μg/ml ampicillin. Clones wereanalyzed by restriction digests and positive clones were sent for DNAsequencing to confirm the sequence of the fusion construct. 1 μl of apositive clone was transformed into 37 μl of DH10B electrocompetent E.coli and streaked on a LB/amp plate. A single colony was picked fromthis streaked plate to start a 250 ml LB/amp culture that was then grownovernight at 37° C. with shaking at 250 rpm. This culture was used togenerate 750 μg of purified DNA using a Qiagen plasmid Maxi kit(Qiagen).

Example 12

[0288] Transfection and Expression of Zcytor17 Soluble ReceptorPolypeptides

[0289] BHK 570 cells (ATCC No. CRL-10314), DG-44 CHO, or other mammaliancells are plated at about 1.2×10⁶ cells/well (6-well plate) in 800 μl ofappropriate serum free (SF) media (e.g., DMEM, Gibco/BRL High Glucose)(Gibco BRL, Gaithersburg, Md.). The cells are transfected withexpression plasmids containing zcytor17CEE, zcytor17CFLG, zcytor17CHISor zcytor17-Fc4 (Example 11), using Lipofectin™ (Gibco BRL), in serumfree (SF) media according to manufacturer's instruction. Single clonesexpressing the soluble receptors are isolated, screened and grown up incell culture media, and purified using standard techniques.

[0290] A. Mammalian Expression of Soluble Human zcytor17CEE Receptor

[0291] BHK 570 cells (ATCC NO: CRL-10314) were plated in T-75 tissueculture flasks and allowed to grow to approximately 50 to 70% confluenceat 37° C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL High Glucose,(Gibco BRL, Gaithersburg, Md.), 5% fetal bovine serum, 1 mM L-glutamine(JRH Biosciences, Lenea, Kans.), 1 mM sodium pyruvate (Gibco BRL)). Thecells were then transfected with the plasmid containing zcytor17CEE(Example 11A) using Lipofectamine™ (Gibco BRL), in serum free (SF) mediaformulation (DMEM, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/mlfetuin, 1% L-glutamine and 1% sodium pyruvate). Ten μg of the plasmidDNA pZp9zyctor17CEE (Example 11A) was diluted into a 15 ml tube to atotal final volume of 500 μl with SF media. 50 μl of Lipofectamine wasmixed with 450 μl of SF medium. The Lipofectamine mix was added to theDNA mix and allowed to incubate approximately 30 minutes at roomtemperature. Four ml of SF media was added to the DNA:Lipofectaminemixture. The cells were rinsed once with 5 ml of SF media, aspirated,and the DNA:Lipofectamine mixture was added. The cells were incubated at37° C. for five hours, and then 5 ml of DMEM/10% FBS media was added.The flask was incubated at 37° C. overnight after which time the cellswere split into the selection media (DMEM/FBS media from above with theaddition of 1 μM methotrexate or 10 μM Methotrexate (Sigma Chemical Co.,St. Louis, Mo.) in 150 mm plates at 1:2, 1:10, and 1:50. Approximately10 days post-transfection, one 150 mm plate of 1 μM methotrexateresistant colonies was trypsinized, the cells were pooled, and one-halfof the cells were replated in 10 μM methotrexate; to further amplifyexpression of the zcytor17CEE protein. A conditioned-media sample fromthis pool of amplified cells was tested for expression levels usingSDS-PAGE and Western analysis.

[0292] B. Mammalian Expression of Soluble Human zcytor17-Fc4 Receptor

[0293] Twenty μg of pEZE-2hzcytor17Fc4 plasmid DNA (Example 11B) waslinearized by restriction digestion with FspI, a restriction enzyme thatcuts once within the pEZE-2 vector and does not disturb genes necessaryfor expression. 200 μg of sheared salmon sperm DNA was added as carrierDNA and then the DNA was precipitated by addition of 0.1 volumes of 3MSodium Acetate pH 5.2 and 2.2 volumes ethanol followed by a 15 minuteice incubation and microcentrifugation at 4° C. The resulting DNA pelletwas washed in 70% ethanol and air dried before being resuspended in 100μl PF CHO non-selection growth media (21 g/L PF CHO Ex Cell 325/200 mML-glutamine (Gibco)/100 mM sodium pyruvate (Gibco)/1×HT Supplement(Gibco). Five million PF CHO passage 43 cells were added to the DNA in600 μl of PF CHO non-selection growth media and then ectroporated in aGene Pulser II Electroporation system (BioRad) using 1070 μF capacitanceand 380 volts using a 0.4 cm gap Gene Pulser (BioRad) electroporationcuvette. The electroporated cells were allowed to recover for 48 hoursin non-selection growth media before selection in −HT media (21 g/L PFCHO Ex Cell 325/200 mM L-glutamine (Gibco)/100 mM sodium pyruvate(Gibco). Cells were selected for 5 days in −HT media before beingpassaged at 5×10⁵ ml into 50 nm MTX selection. Cells selected at 50 nmMTX were seeded at 6×10⁵ ml in a shake flask to generate conditionedmedia. The resulting 72 hour conditioned media was analyzed by probingwestern blots with an antibody generated against human Ig. The cellsproduced hzcytor17/Fc4 protein at approximately 1 mg/L.

[0294] C. Larger-scale Mammalian Expression of Soluble Humanzcytor17-Fc4 Receptor

[0295] Two hundred μg of pEZE-2hzcytor17Fc4 plasmid DNA (Example 11B)was linearized by restriction digestion with FspI, a restriction enzymethat cuts once within the pEZE-2 vector and does not disturb genesnecessary for expression. 200 μg of CHO genomic DNA (prepared in-house)was added as carrier DNA and then the DNA was precipitated by additionof 0.1 volumes of 3M Sodium Acetate pH 5.2 and 2.5 volumes ethanolfollowed by microcentrifugation at Room temperature. Five replicate DNApellets were made and transformed. The resulting DNA pellet was washedin 70% ethanol and air dried before being resuspended in 100 μl PF CHOnon-selection growth media (21 g/L PF CHO Ex Cell 325/200 mM L-glutamine(Gibco)/100 mM sodium pyruvate (Gibco)/1×HT Supplement (Gibco). Tenmillion PF CHO cells were added to the DNA in 600 μl of PF CHOnon-selection growth media and then electroporated in a Gene Pulser IIElectroporation system (BioRad) using 950 μF capacitance and 300 voltsusing a 0.4 cm gap Gene Pulser (BioRad) electroporation cuvette. Theelectroporated cells were pooled and put directly into selection in −HTmedia (21 g/L PF CHO Ex Cell 325/200 mM L-glutamine (Gibco)/100 mMsodium pyruvate (Gibco). Cells were selected for 14 days in -HT mediabefore being passaged at 4×10⁵/ml into 50 nm MTX selection. Cells wereamplified to 200 nM MTX and then to 1 uM MTX. The −HT, 5 nM, and 1 uMpools were seeded at 1×10⁶ c/ml for 48 hours, and the resultingconditioned media was analyzed by probing western blots with an antibodygenerated against human Ig.

[0296] C. Transient Mammalian Expression and Purification of SolubleHuman zcytor17-Fc4 Receptor

[0297] pEZE-2hzcytor17Fc4 plasmid DNA (Example 11B) was introduced into40 maxi plates of BHK cells using Lipofectamine (Gibco BRL) as describedherein and in manufacturer's instructions. Cells were allowed to recoverovernight, then were rinsed and refed with serum-free medium (SL7V4,made in-house). After 72 hours, the media was collected and filtered,and cells were refed with serum-free medium. After 72 hours, the mediawas again collected and filtered.

[0298] The serum-free conditioned media (2×1.5 L batches) fromtransiently transfected BHK cells was pumped over a 1.5 ml ProteinA-agarose column in 20 mM Tris, pH 7.5, 0.5 M NaCl. The column waswashed extensively with this buffer and then the bound protein waseluted with 1 ml of 0.2 M glycine, pH 2.5, 0.5 M NaCl. The elutedprotein was collected into 0.1 ml of 2 M Tris, pH 8.5.

[0299] Aliquots were collected for SDS-polyacrylamide gelelectrophoresis and the bulk zcytor17-Fc was dialyzed overnight againstPBS. The soluble receptor was sterile filtered and placed in aliquots at−80° C.

Example 13

[0300] Expression of zcytor17 Soluble Receptor in E. coli

[0301] A. Construction of Expression Vector pCZR225 that Expresseshuzcytor17/MBP-6H Fusion Polypeptide

[0302] An expression plasmid containing a polynucleotide encoding azcytor17 soluble receptor fused C-terminally to maltose binding protein(MBP) was constructed via homologous recombination. The fusionpolypeptide contains an N-terminal approximately 388 amino acid MBPportion fused to any of the zcytor17 soluble receptors described herein.A fragment of zcytor17 cDNA (SEQ ID NO:1, SEQ ID NO:45, SEQ ID NO:17 orSEQ ID NO:21) was isolated using PCR as described herein. Two primerswere used in the production of the zcytor17 fragment in a standard PCRreaction: (1) one containing about 40 bp of the vector flanking sequenceand about 25 bp corresponding to the amino terminus of the zcytor17, and(2) another containing about 40 bp of the 3′ end corresponding to theflanking vector sequence and about 25 bp corresponding to the carboxylterminus of the zcytor17. Two μl of the 100 μl PCR reaction was run on a1.0% agarose gel with 1×TBE buffer for analysis, and the expectedapproximately fragment was seen. The remaining PCR reaction was combinedwith the second PCR tube and precipitated with 400 μl of absoluteethanol. The precipitated DNA used for recombining into the Sma1 cutrecipient vector pTAP170 to produce the construct encoding theMBP-zcytor17 fusion, as described below.

[0303] Plasmid pTAP170 was derived from the plasmids pRS316 and pMAL-c2.The plasmid pRS316 was a Saccharomyces cerevisiae shuttle vector (HieterP. and Sikorski, R., Genetics 122:19-27, 1989). pMAL-C2 (NEB) was an E.coli expression plasmid. It carries the tac promoter driving MalE (geneencoding MBP) followed by a His tag, a thrombin cleavage site, a cloningsite, and the rrnB terminator. The vector pTAP98 was constructed usingyeast homologous recombination. 100 ng of EcoR1 cut pMAL-c2 wasrecombined with 1 μg Pvu1 cut pRS316, 1 μg linker, and 1 μg Sca1/EcoR1cut pRS316 were combined in a PCR reaction. PCR products wereconcentrated via 100% ethanol precipitation.

[0304] Competent yeast cells (S. cerevisiae) were combined with about 10μl of a mixture containing approximately 1 μg of the zcytor17 receptorPCR product above, and 100 ng of SmaI digested pTAP98 vector, andelectroporated using standard methods and plated onto URA-D plates andincubated at 30° C.

[0305] After about 48 hours, the Ura+ yeast transformants from a singleplate were picked, DNA was isolated, and transformed intoelectrocompetent E. coli cells (e.g., MC1061, Casadaban et. al. J. Mol.Biol. 138, 179-207), and plated on MM/CA+AMP 100 mg/L plates (Pryor andLeiting, Protein Expression and Purification 10:309-319, 1997), usingstandard procedures. Cells were grown in MM/CA with 100 μg/ml Ampicillinfor two hours, shaking, at 37° C. 1 ml of the culture was induced with 1mM IPTG. 2-4 hours later the 250 μl of each culture was mixed with 250μl acid washed glass beads and 250 μl Thorner buffer with 5% βME and dye(8M urea, 100 mM Tris pH7.0, 10% glycerol, 2 mM EDTA, 5% SDS). Sampleswere vortexed for one minute and heated to 65° C. for 10 minutes. 20 μlwere loaded per lane on a 4%-12% PAGE gel (NOVEX). Gels were run in1×MES buffer. The positive clones were designated pCZR225 and subjectedto sequence analysis.

[0306] One microliter of sequencing DNA was used to transform strainBL21. The cells were electropulsed at 2.0 kV, 25 μF and 400 ohms.Following electroporation, 0.6 ml MM/CA with 100 mg/L Ampicillin. Cellswere grown in MM/CA and induced with ITPG as described above., Thepositive clones were used to grow up for protein purification of thehuzcytor17/MBP-6H fusion protein using standard techniques.

[0307] B. Purification of huzcytor17/MBP-6H Soluble Receptor from E.coliFermentation

[0308] Unless otherwise noted, all operations were carried out at 4° C.The following procedure was used for purifying huzcytor17/MBP-6H solublereceptor polypeptide. E. coli cells containing the pCZR225 construct andexpressing huzcytor17/MBP-6H soluble receptor (Example 13A) were grownup in SuperBroth II (12 g/L Casien, 24 g/L Yeast Extract, 11.4 g/Ldi-potassium phosphate, 1.7 g/L Mono-potassium phosphate; BectonDickenson, Cockeysville, Md.), and frozen in 0.5% glycerol. Twenty gramsof the frozen cells in SuperBroth II+Glycerol were used to purify theprotein. The frozen cells were thawed and diluted 1:10 in a proteaseinhibitor solution (Extraction buffer) prior to lysing the cells andreleasing the huzcytor17MBP-6H soluble receptor protein. The dilutedcells contained final concentrations of 20 mM Tris (J T Baker,Philipsburg, N.J.) 100 mM Sodium Chloride (NaCl, Mallinkrodt, Paris,Ky.), 0.5 mM pheynlmethylsulfonyl fluoride (PMSF, Sigma Chemical Co.,St. Louis, Mo.), 2 μg/nm Leupeptin (Fluka, Switzerland), and 2 μg/mlAprotinin (Sigma). A French Press cell breaking system (Constant SystemsLtd., Warwick, UK) with temperature of −7 to −10° C. and 30K PSI wasused to lyse the cells. The diluted cells were checked for breakage byA₆₀₀ readings before and after the French Press. The lysed cells werecentrifuged @ 18,000 G for 45 minutes to remove the broken cell debris,and the supernatant used to purify the protein.

[0309] A 25 ml column of Amylose resin (New England Biolabs, Beverly,Mass.) (prepared as described below) was poured in a Bio-Rad, 2.5 cmD×10 cm H glass column. The column was packed and equilibrated bygravity with 10 column volumes (CVs) of Amylose Equilibration buffer (20mM Tris, 100 mM NaCl, pH 8.0). The supernatant was batch loaded to theAmylose resin and was rocked overnight. The resin was poured back intothe column and was washed with 10 CV's of Amylose Equilibration bufferby gravity. The column was washed for 10 CVs with Amylose equilibrationbuffer, then eluted with ˜2 CV of Amylose equilibration buffer+10 mMMaltose (Fluka Biochemical, Switzerland) by gravity. 5 ml fractions werecollected over the entire chromatography and absorbance at 280 and 320mM were read. The Amylose column was regenerated with 1 CV of distilledH₂O, 5 CVs of 0.1% (w/v) SDS (Sigma), 5 CVs of distilled H₂O, and then 5CVs of Amylose equilibration buffer.

[0310] Fractions of interest were pooled and dialyzed in a Slide-A-Lyzer(Pierce) with 4×4 L PBS pH 7.4 (Sigma) to remove low molecular weightcontaminants, buffer exchange and desalt. After the changes of PBS, thematerial harvested represented the purified huzcytor17/MBP-6Hpolypeptide. The purified huzcytor17/MBP-6H polypeptide was analyzed viaSDS-PAGE Coomassie staining and Western blot analyses with theanti-rabbit HRP conjugated antibody (Rockland, Gilbertsville, Pa.). Theconcentration of the huzcytor17/MBP-6H polypeptide was 1.92 mg/ml asdetermined by BCA analysis.

[0311] Purified huzcytor17/MBP-6H polypeptide was prepared for injectioninto rabbits and sent to R & R Research and Development (Stanwood,Wash.) for antibody production. Rabbits were injected to produce antianti-huzcytor17/MBP-6H serum (Example 15, below).

Example 14

[0312] Zcytor17 Soluble Receptor Polyclonal Antibodies

[0313] Polyclonal antibodies were prepared by immunizing 2 female NewZealand white rabbits with the purified huzcytor17/MBP-6H polypeptide(Example 13). The rabbits are each given an initial intraperitoneal (IP)injection of 200 μg of purified protein in Complete Freund's Adjuvant(Pierce, Rockford, Ill.) followed by booster IP injections of 100 ugpurified protein in Incomplete Freund's Adjuvant every three weeks.Seven to ten days after the administration of the third boosterinjection, the animals are bled and the serum is collected. The rabbitsare then boosted and bled every three weeks.

[0314] The zcytor17-specific polyclonal antibodies are affinity purifiedfrom the rabbit serum using an CNBr-SEPHAROSE 4B protein column(Pharmacia LKB) that is prepared using about 10 mg of the purifiedhuzcytor17/MBP-6H polypeptide per gram CNBr-SEPHAROSE, followed by 20×dialysis in PBS overnight. Zcytor17-specific antibodies arecharacterized by an ELISA titer check using 1 μg/ml of the appropriateprotein antigen as an antibody target. The lower limit of detection(LLD) of the rabbit anti-zcytor17 affinity purified antibodies isdetermined using standard methods.

Example 15

[0315] Zcytor17 Receptor Monoclonal Antibodies

[0316] Zcytor17 soluble receptor Monoclonal antibodies are prepared byimmunizing female BalbC mice with the purified recombinant solublezcytor17 proteins described herein. The mice are each given an initialintraperitoneal (IP) injection of 20 ug of purified protein in CompleteFreund's Adjuvant (Pierce, Rockford, Ill.) followed by booster IPinjections of 10 ug purified protein in Incomplete Freund's Adjuvantevery two weeks. Seven to ten days after the administration of the thirdbooster injection, the animals are bled and the serum is collected, andantibody titer assessed.

[0317] Splenocytes are harvested from high-titer mice and fused tomurine SP2/0 myeloma cells using PEG 1500 (Boerhinger Mannheim, UK)using a 4:1 fusion ratio of splenocytes to myeloma cells (Antibodies: ALaboratory Manual, E. Harlow and D. Lane, Cold Spring Harbor Press).Following 10 days growth post-fusion, specific antibody-producinghybridomas are identified by ELISA using purified recombinant zcytor17soluble receptor protein (Example 6C) as an antibody target and by FACSusing Baf3 cells expressing the zcytor17 sequence (Example 8) as anantibody target. The resulting hybridomas positive by both methods arecloned three times by limiting dilution.

Example 16

[0318] Assessing Zcytor17 Receptor Heterodimerization Using ORIGEN Assay

[0319] Soluble zcytor17 receptor zcytor17CFLAG (Example 11), or gp130(Hibi, M. et al., Cell 63:1149-1157, 1990) are biotinylated by reactionwith a five-fold molar excess of sulfo-NHS-LC-Biotin (Pierce, Inc.,Rockford, Ill.) according to the manufacturer's protocol. Solublezcytor17 receptor and another soluble receptor subunit, for example,soluble gp130, LIF, IL-12, WSX-1, L-7Rα (sIL-7Rα) or IL-2 receptorγ(sIL-2Rγ) (R&D Systems, Minneapolis, Minn.), or soluble zalpha 11receptor (IL-21R; commonly owned U.S. patent application Ser. No.09/404,641) are labeled with a five fold molar excess of Ru-BPY-NHS(Igen, Inc., Gaithersburg, Md.) according to manufacturer's protocol.The biotinylated and Ru-BPY-NHS-labeled forms of the soluble zcytor17receptor can be respectively designated Bio-zcytor17 receptor andRu-zcytor17; the biotinylated and Ru-BPY-NHS-labeled forms of the othersoluble receptor subunit can be similarly designated. Assays can becarried out using conditioned media from cells expressing a ligand thatbinds zcytor17 heterodimeric receptors, or using purified ligands.Preferred ligands are those that can bind class 1 heterodimeric cytokinereceptors such as, gp130, LIF, IL-12, IL-2, IL4, IL-7, IL-9, IL-15,zalpha11 Ligand (IL-21) (commonly owned U.S. patent application Ser. No.09/522,217), TSLP (Levine, S D et al., ibid.; Isaksen, D E et al.,ibid.; Ray, R J et al., ibid.; Friend, S L et al., ibid.).

[0320] For initial receptor binding characterization a panel ofcytokines or conditioned medium are tested to determine whether they canmediate homodimerization of zcytor17 receptor and if they can mediatethe heterodimerization of zcytor17 receptor with the soluble receptorsubunits described above. To do this, 50 μl of conditioned media orTBS-B containing purified cytokine, is combined with 50 μl of TBS-B (20mM Tris, 150 mM NaCl, 1 mg/ml BSA, pH 7.2) containing e.g., 400 ng/ml ofRu-zcytor17 receptor and Bio-zcytor17, or 400 ng/ml of Ru-zcytor17receptor and e.g., Bio-gp130, or 400 ng/ml of e.g., Ru-IL2Rγ andBio-zcytor17. Following incubation for one hour at room temperature, 30μg of streptavidin coated, 2.8 mm magnetic beads (Dynal, Inc., Oslo,Norway) are added and the reaction incubated an additional hour at roomtemperature. 200 μl ORIGEN assay buffer (Igen, Inc., Gaithersburg, Md.)is then added and the extent of receptor association measured using anM8 ORIGEN analyzer (Igen, Inc.).

Example 17

[0321] Construct for Generating a zcytor17 Receptor Heterodimer

[0322] A vector expressing a secreted human zcytor17 heterodimer isconstructed. In this construct, the extracellular cytokine-bindingdomain of zcytor17 is fused to the heavy chain of IgG gamma 1 (IgGγ1)(SEQ ID NO:37 and SEQ ID NO:38), while the extracellular portion of theheteromeric cytokine receptor subunit (E.g., an gp130, LIF, IL-12,WSX-1, or IL-2 receptor component (IL-2Rα, IL-2Rβ, IL-2Rγ), anIL-4/IL-13 receptor family receptor components (IL-4Rα, IL-13Rα,IL-13Rα′), interleukin receptor subunits (e.g., IL-15 Rα, IL-7Rα,IL-9Rα; or zalpha11 receptor (IL-21R)) is fused to a human kappa lightchain (human κ light chain) (SEQ ID NO:39 and SEQ ID NO:40).

[0323] A. Construction of IgG Gamma 1 and Human κ Light Chain FusionVectors

[0324] The heavy chain of IgGγ1 (SEQ ID NO:37) is cloned into theZem229R mammalian expression vector (ATCC deposit No. 69447) such thatany desired cytokine receptor extracellular domain having a 5′ EcoRI and3′ NheI site can be cloned in resulting in an N-terminal extracellulardomain-C-terminal IgGγ1 fusion. The IgGγ1 fragment used in thisconstruct is made by using PCR to isolate the IgGγ1 sequence from aClontech hFetal Liver cDNA library as a template. PCR products arepurified using methods described herein and digested with MluI and EcoRI(Boerhinger-Mannheim), ethanol precipitated and ligated with oligosZC11,440 (SEQ ID NO:41) and ZC11,441 (SEQ ID NO:42), which comprise anMluI/EcoRI linker, into Zem229R previously digested with and EcoRI usingstandard molecular biology techniques disclosed herein.

[0325] The human κ light chain (SEQ ID NO:39) is cloned in the Zem228Rmammalian expression vector (ATCC deposit No. 69446) such that anydesired cytokine receptor extracellular domain having a 5′ EcoRI siteand a 3′ KpnI site can be cloned in resulting in a N-terminal cytokineextracellular domain-C-terminal human κ light chain fusion. As a KpnIsite is located within the human κ light chain sequence (cleaved by theKpnI enzyme after nucleotide 62 in SEQ ID NO:39), a special primer isdesigned to clone the 3′ end of the desired extracellular domain of acytokine receptor into this KpnI site: The primer is designed so thatthe resulting PCR product contains the desired cytokine receptorextracellular domain with a segment of the human κ light chain up to theKpnI site (SEQ ID NO:39). This primer preferably comprises a portion ofat least 10 nucleotides of the 3′ end of the desired cytokine receptorextracellular domain fused in frame 5′ to SEQ ID NO:39. The human κlight chain fragment used in this construct is made by using PCR toisolate the human κ light chain sequence from the same Clontech humanFetal Liver cDNA library used above. PCR products are purified usingmethods described herein and digested with MluI and EcoRI(Boerhinger-Mannheim), ethanol precipitated and ligated with theMluI/EcoRI linker described above, into Zem228R previously digested withand EcoRI using standard molecular biology techniques disclosed herein.

[0326] B. Insertion of zcytor17 Receptor or Heterodimeric SubunitExtracellular Domains into Fusion Vector Constructs

[0327] Using the construction vectors above, a construct having zcytor17fused to IgGγ1 is made. This construction is done by PCRing theextracellular domain or cytokine-binding domain of zcytor17 receptordescribed herein from a prostate cDNA library (Clontech) or activatedlymphocyte cDNA library using standard methods (E.g., Example 7), andoligos that provide EcoRI and NheI restriction sites. The resulting PCRproduct is digested with EcoRI and NheI, gel purified, as describedherein, and ligated into a previously EcoRI and NheI digested andband-purified Zem229R/IgGγ1 described above. The resulting vector issequenced to confirm that the zcytor17/IgG gamma 1 fusion (zcytor17/Ch1IgG) is correct.

[0328] A separate construct having a heterodimeric cytokine receptorsubunit extracellular domain fused to κ light is also constructed asabove. The cytokine receptor/human κ light chain construction isperformed as above by PCRing from, e.g., a lymphocyte cDNA library(Clontech) using standard methods, and oligos that provide EcoRI andKpnI restriction sites. The resulting PCR product is digested with EcoRIand KpnI and then ligating this product into a previously EcoRI and KpnIdigested and band-purified Zem228R/human κ light chain vector describedabove. The resulting vector is sequenced to confirm that the cytokinereceptor subunit/human κ light chain fusion is correct.

[0329] D. Co-expression of the zcytor17 and Heterodimeric CytokineReceptor Subunit Extracellular Domain

[0330] Approximately 15 μg of each of vectors above, are co-transfectedinto mammalian cells, e.g., BHK-570 cells (ATCC No. CRL-10314) usingLipofectaminePlus™ reagent (Gibco/BRL), as per manufacturer'sinstructions. The transfected cells are selected for 10 days in DMEM+5%FBS (Gibco/BRL) containing 1 μM of methotrexate (MTX) (Sigma, St. Louis,Mo.) and 0.5 mg/ml G418 (Gibco/BRL) for 10 days. The resulting pool oftransfectants is selected again in 10 μm of MTX and 0.5 mg/ml G418 for10 days.

[0331] The resulting pool of doubly selected cells is used to generateprotein. Three Factories (Nunc, Denmark) of this pool are used togenerate 10 L of serum free conditioned medium. This conditioned mediais passed over a 1 ml protein-A column and eluted in about 10, 750microliter fractions. The fractions having the highest proteinconcentration are pooled and dialyzed (10 kD MW cutoff) against PBS.Finally the dialyzed material is submitted for amino acid analysis (AAA)using routine methods.

Example 18

[0332] Determination of Receptor Subunits that Heterodimerize orMultimerize with zcytor17 Receptor

[0333] Using standard methods described herein, The BaF3/MPL-zcytor17chimera cells (Example 6) are transfected with an additionalheterodimeric cytokine receptor subunit serve as a bioassay cell line tomeasure signal transduction response of heterodimeric zcytor17 receptorcomplexes to the luciferase reporter in the presence of TPO (Example 6).Transfection of the BaF3/MPL-zcytor17 cell line with and additionalMPL-class I cytokine receptor fusion that signals in the presence of theTPO ligand, determines which heterodimeric cytokine receptor subunitsare required for zcytor17 receptor signaling. Use of MPL-receptorfusions for this purpose alleviates the requirement for the presence ofa natural ligand for the zcytor17 receptor.

[0334] MPL-class I cytokine receptor fusions are made as per Example 5using the extracellular domain and transmembrane domains of the MPLreceptor and the intracellular signaling domain of the desired class Icytokine receptor. The BaF3/MPL-zcytor17 bioassay cell lineco-transfected with an individual MPL-class I cytokine receptor fusionsas per Example 6 to form a BaF3/MPL-zcytor17/MPL-class I cytokinereceptor cell line. Receptor complexes include but are not limited tozcytor17 receptor in combination with an MPL-cytokine receptor fusioncomprising a gp130, LIF, IL-12, or WSX-1 component, or one or more ofthe IL-2 receptor components (IL-2Rα, IL-2Rβ, IL-2Rγ), zcytor17 receptorwith one or more of the IL-4/IL-13 receptor family receptor components(L-4Rα, IL-13Rα, IL-13Rα′), as well as other Interleukin receptors(e.g., IL-15 Rα, IL-7Rα, IL-9Rα, IL-21R (Zalpha11 receptor)). Eachindependent receptor complex cell line is then assayed in the presenceof TPO (example 6) and proliferation measured using routine methods(e.g., Alamar Blue assay as described in Example 6). TheBaF3/MPL-zcytor17 bioassay cell line serves as a control for thebackground activity, and is thus used as a baseline to compare signalingby the various receptor complex combinations. Moreover, assay byluciferase reporter assay (activation of transcription of a reportergene) can also be used as a way to measure signaling regardless ofinduction of proliferation. In addition, a BaF3/MPL-class I cytokinereceptor cell line can be constructed to control for MPL-class Icytokine receptor homodimerization effects for those class I cytokinereceptors known to signal upon homodimerization. The TPO in the presenceof the correct receptor complex, is expected to increase proliferationof the BaF3/MPL-zcytor17/MPL-class I cytokine receptor cell lineapproximately 5 fold over background or greater in the presence of TPO.

Example 19

[0335] Reconstitution of zcytor17 Receptor in Vitro

[0336] To identify components involved in the zcytor17-signalingcomplex, receptor reconstitution studies are performed as follows. Forexample, BHK 570 cells (ATCC No. CRL-10314) transfected, using standardmethods described herein, with a luciferase reporter mammalianexpression vector plasmid serve as a bioassay cell line to measuresignal transduction response from a transfected zcytor17 receptorcomplex to the luciferase reporter in the presence of zcytor17 Ligand.BHK cells would be used in the event that BHK cells do not endogenouslyexpress the zcytor17 receptor. Other cell lines can be used. Anexemplary luciferase reporter mammalian expression vector is the KZ134plasmid which is constructed with complementary oligonucleotidesZC12,749 (SEQ ID NO:43) and ZC12,748 (SEQ ID NO:44) that contain STATtranscription factor binding elements from 4 genes. A modified c-fos Sisinducible element (m67SIE, or hSIE) (Sadowski, H. et al., Science261:1739-1744, 1993), the p21 SIE1 from the p21 WAF1 gene (Chin, Y. etal., Science 272:719-722, 1996), the mammary gland response element ofthe β-casein gene (Schmitt-Ney, M. et al., Mol. Cell. Biol.11:3745-3755, 1991), and a STAT inducible element of the Fcg RI gene,(Seidel, H. et al., Proc. Natl. Acad. Sci. 92:3041-3045, 1995). Theseoligonucleotides contain Asp718-XhoI compatible ends and are ligated,using standard methods, into a recipient firefly luciferase reportervector with a c-Fos promoter (Poulsen, L. K. et al., J. Biol. Chem.273:6229-6232, 1998) digested with the same enzymes and containing aneomycin selectable marker. The KZ134 plasmid is used to stablytransfect BHK, or BaF3 cells, using standard transfection and selectionmethods, to make a BHK/KZ134 or BaF3/KZ134 cell line respectively.

[0337] The bioassay cell line is transfected with zcytor17 receptoralone, or co-transfected with zcytor17 receptor along with one of avariety of other known receptor subunits. Receptor complexes include butare not limited to zcytor17 receptor only, various combinations ofzcytor17 receptor with gp130, LIF, IL-12, or WSX-1 receptor subunits, orone or more of the IL-2 receptor components (IL-2Rα, IL-2Rβ, IL-2Rγ),zcytor17 receptor with one or more of the IL-4/IL-13 receptor familyreceptor components (IL-4Rα, IL-13Rα, IL-13Rα′), as well as otherInterleukin receptors (e.g., IL-15 Rα, IL-7Rα, IL-9Rα, IL-21R (zalpha11)). Each independent receptor complex cell line is then assayed in thepresence of cytokine-conditioned media or purified cytokines andluciferase activity measured using routine methods. The untransfectedbioassay cell line serves as a control for the background luciferaseactivity, and is thus used as a baseline to compare signaling by thevarious receptor complex combinations. The conditioned medium orcytokine that binds the zcytor17 receptor in the presence of the correctreceptor complex, is expected to give a luciferase readout ofapproximately 5 fold over background or greater.

[0338] As an alternative, a similar assay can be performed wherein theBaf3/zcytor17-mpl and Baf3/zcytor17 (Example 10) cell lines areco-transfected as described above and proliferation measured.

Example 20

[0339] Construction of BaF3 Cells Expressing Full-Length Zcytor17

[0340] BaF3, an interleukin-3 (IL-3) dependent prelymphoid cell linederived from murine bone marrow (Palacios and Steinmetz, Cell 41:727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6.: 4133-4135,1986), was maintained in complete media (RPMI medium (JRH BioscienceInc., Lenexa, Kans.) supplemented with 10% heat-inactivated fetal calfserum, 2 ng/ml murine IL-3 (mIL-3) (R+D, Minneapolis, Minn.), 2 mML-glutamine (Gibco-BRL), and 1 mM Sodium Pyruvate (Gibco-BRL).

[0341] BaF3 cells for electroporation were washed twice in PBS, pH 7.2(Gibco-BRL) and then resuspended in PBS and a cell density of 1cells/ml. One ml of resuspended BaF3 cells was mixed with 30 μg of thepZP7PX/zcytor17 plasmid DNA and transferred to separate disposableelectroporation chambers (Gibco-BRL). The cells were then given 2 serialshocks (800 lFad/300V.; 1180 lFad/300V.) delivered by an electroporationapparatus (CELL-PORATOR™; Gibco-BRL). The electroporated cells were thentransferred to 20 mls of complete media and placed in an incubator for48 hours (37° C., 5% CO₂). The cells were then spun down and resuspendedin 20 mls of complete media containing 2 μg/ml Puromycin (ClonTech)Selection in a T75 flask to isolate the puromycin resistant pool. Clonallines of the transfected BaF3 cells, hereinafter called BaF3/zcytor17cells, were isolated as described below.

[0342] BaF3/zcytor17 cells were counted in a hemocytometer, and platedat 1 cell/well, 0.5 cell/well, 0.1 cell/well, and 0.01 cell/well, in avolume of 100 μl/well in complete media containing 2 μg/ml Puromycin. 15clones were scaled up to T75 flasks, and 5 clones were assayed for RNAproduction. Cells were washed once with PBS and counted with ahemocytometer. 5×10⁶ cells were spun down and the media removed.Untransfected BaF3 cells were also counted and pelleted. Four of thepellets were frozen at −80° C. overnight. RNA was isolated using theS.N.A.P.™ Total RNA Isolation Kit (Invitrogen) as per manufacturer'sinstructions, and total RNA yield was determined by spectrophotometer.The amount of zcytor17 RNA was then determined by RT-PCR using theRT-PCR Kit (Stratagene) as per manufacturer's instructions, usingzcytor17-specific primers ZC29,180 (SEQ ID NO:72) and ZC29,122 (SEQ IDNO:65). One clone that gave a strong band of zcytor17 RNA was selectedto use in BaF3 Alamar Blue proliferation assays (Example 6) to testpotential ligands.

Example 21

[0343] Cloning of Mouse zcytoR17 from a Mouse Testes cDNA Library

[0344] A mouse testes cDNA library was screened for a full-length cloneof mouse zcytoR17. The library was plated at 65,500 cfu/plate on 24LB+Amp plates. Filter lifts were prepared using Hybond N(Amersham-Pharmacia Biotech, Inc., Piscataway, N.J.) on a total ofapproximately 1.6 million colonies. The filters were marked with a hotneedle for orientation and then denatured for 6 minutes in 0.5 M NaOHand 1.5 M Tris-HCl, pH 7.2. The filters were then neutralized in 1.5 MNaCl and 0.5 M Tris-HCl, pH 7.2 for 6 minutes. The DNA was affixed tothe filters using a UV crosslinker (Stratalinker®, Stratagene, La Jolla,Calif.) at 1200 joules. The filters were then left to dry overnight atroom temperature.

[0345] The next day, the filters were pre-washed at 65° C. in pre-washbuffer consisting of 0.25×SSC, 0.25% SDS and 1 mM EDTA. Cell debris wasmanually removed using Kimwipes® (Kimberly-Clark) and the solution waschanged 3 times over a period of 1 hour. Filters were air dried andstored at room temperature until needed. The filters were thenprehybridized for approximately 3 hours at 63° C. in 20 ml ofExpressHyb™Hybridization Solution (Clontech, Palo Alto, Calif.).

[0346] Probe B (Example 3C) was generated by PCR from human zcytoR17template using oligonucleotide primers ZC27,895 (SEQ ID NO:64) andZC28,917 (SEQ ID NO:73) and was radioactively labeled with ³²P using acommercially available kit (Megaprime DNA Labeling System; AmershamPharmacia Biotech, Piscataway, N.J.) according to the manufacturer'sinstructions. The probe was purified using a Stratagene™ push column(NucTrap® column; Stratagene, La Jolla, Calif.). The probe was denaturedat 100° C. for 15 min and added to ExpressHyb™. Filters were hybridizedin 15 ml hybridizing solution containing 1.6×10⁶ cpm/ml of probe at 63°C. overnight. Filters were washed at 55° C. in 2×SSC, 0.1% SDS and 1 mMEDTA and exposed to X-ray film at −80° C. for 4½ days. Thirteenpositives were picked from the plates as plugs and placed in 1 ml LB+ampin 1.7 ml tubes. Tubes were placed at 4° C. overnight. These 13positives were subjected to two further rounds of purification. Thetertiary plates were outgrown at 37° C. after filter lifts were takenand single colonies were picked and sent to sequencing. Three of thesewere determined to contain sequence of the mouse ortholog of zcytoR17.

[0347] In addition, a PCR product was generated using CTLL-2 cDNA as atemplate and oligonucleotides ZC38,239 (SEQ ID NO:88) and ZC38,245 (SEQID NO:89) as primers. CTLL-2 is a mouse cytotoxic T lymphocyte cell line(ATCC No. TIB-214). This PCR reaction was run as follows: 1 cycle at 95°C. for 1 minute, 30 cycles at 95° C. for 15 seconds, 68° C. for 3minutes, then 68° C. for 10 minutes; 4° C. soak. The PCR reaction usedapproximately 0.5 ng. of cDNA, 20 pmoles of each oligonucleotide, and 1μl. of Advantage II polymerase mix (ClonTech). About 6% of the PCRproduct was used as a template in a new PCR reaction, as above, exceptwith oligonucleotides ZC38,239 (SEQ ID NO:88) and ZC38,238 (SEQ IDNO:90). This PCR reaction was run as follows: 30 cycles at 94° C. for 45seconds, 65° C. for 45 seconds, 72° C. for 1 minute, then 72° C. for 7minutes; 10° C. soak. Most of the PCR reaction was loaded on a 1.0%agarose gel and the predominant band at approximately 360 bp wasexcised, the DNA fragment was eluted, and DNA sequencing was performed.

[0348] The sequence of the mouse zcytor17 polynucleotide is shown in SEQID NO:56 and the corresponding amino acid sequence shown in SEQ IDNO:57. In addition, a truncated soluble form of the mouse zcytor17polynucleotide is shown in SEQ ID NO:92 and the corresponding amino acidsequence shown in SEQ ID NO:93.

Example 22

[0349] Tissue Distribution of Human zcytor17 in Tissue Panels Using PCR

[0350] A panel of cDNAs from murine tissues was screened for mousezcytor17 expression using PCR. The panel was made in-house and contained94 marathon cDNA and cDNA samples from various normal and cancerousmurine tissues and cell lines are shown in Table 7, below. The cDNAscame from in-house libraries or marathon cDNAs from in-house RNA preps,Clontech RNA, or Invitrogen RNA. The mouse marathon cDNAs were madeusing the marathon-Ready™ kit (Clontech, Palo Alto, Calif.) and QCtested with mouse transferrin receptor primers ZC10,651 (SEQ ID NO:79)and ZC10,565 (SEQ ID NO:80) and then diluted based on the intensity ofthe transferrin band. To assure quality of the amplified library samplesin the panel, three tests for quality control (QC) were run: (1) Toassess the RNA quality used for the libraries, the in-house cDNAs weretested for average insert size by PCR with vector oligos that werespecific for the vector sequences for an individual cDNA library; (2)Standardization of the concentration of the cDNA in panel samples wasachieved using standard PCR methods to amplify full length alpha tubulinor G3PDH cDNA using a 5′ vector oligo: ZC14,063 (SEQ ID NO:7) and 3′alpha tubulin specific oligo primer ZC17,574 (SEQ ID NO:26) or 3′ G3PDHspecific oligo primer ZC17,600 (SEQ ID NO:27); and (3) a sample was sentto sequencing to check for possible ribosomal or mitochondrial DNAcontamination. The panel was set up in a 96-well format that included amouse genomic DNA (Clontech, Palo Alto, Calif.) positive control sample.Each well contained approximately 0.2-100 pg/μl of cDNA. The PCR was setup using oligos ZC38,065 (SEQ ID NO:77) and ZC38,068 (SEQ ID NO:78),TaKaRa Ex Taq™ (TAKARA Shuzo Co LTD, Biomedicals Group, Japan), andRediload dye (Research Genetics, Inc., Huntsville, Ala.). Theamplification was carried out as follows: 1 cycle at 94° C. for 5minutes; 5 cycles of 94° C. for 30 seconds, 68° C. for 30 seconds; 35cycles of 94° C. for 30 seconds, 56° C. for 30 seconds and 72° C. for 30seconds, followed by 1 cycle at 72° C. for 5 minutes. About 10 μl of thePCR reaction product was subjected to standard Agarose gelelectrophoresis using a 4% agarose gel. The correct predicted DNAfragment size was observed in brain, CD90+ cells, dendritic, embryo,MEWt#2, Tuvak-prostate cell line, salivary gland, skin and testis.

[0351] The DNA fragment for skin and testis were excised and purifiedusing a Gel Extraction Kit (Qiagen, Chatsworth, Calif.) according tomanufacturer's instructions. Fragments were confirmed by sequencing toshow that they were indeed mouse zcytor17. TABLE 7 Tissue/Cell line#samples Tissue/Cell line #samples 229 1 7F2 1 Adipocytes-Amplified 1aTC1.9 1 Brain 4 CCC4 1 CD90+ Amplified 1 OC10B 1 Dentritic 1 Embyro 1Heart 2 Kidney 3 Liver 2 Lung 2 MEWt#2 1 P388D1 1 Pancreas 1 Placenta 2Jakotay-Prostate Cell Line 1 Nelix-Prostate Cell Line 1 Paris-ProstateCell Line 1 Torres-Prostate Cell Line 1 Tuvak-Prostate Cell Line 1Salivary Gland 2 Skeletal Muscle 1 Skin 2 Small Intestine 1 SmoothMuscle 2 Spleen 2 Stomach 1 Testis 3 Thymus 1

Example 23

[0352] Zcytor17 Expression in Various Tissues Using Real-TimeQuantitative RT/PCR

[0353] A. Primers and Probes for Quantitative RT-PCR

[0354] Real-time quantitative RT-PCR using the ABI PRISM 7700 SequenceDetection System (PE Applied Biosystems, Inc., Foster City, Calif.) hasbeen previously described (See, Heid, C. A. et al., Genome Research6:986-994, 1996; Gibson, U. E. M. et al., Genome Research 6:995-1001,1996; Sundaresan, S. et al., Endocrinology 139:4756-4764, 1998. Thismethod incorporates use of a gene specific probe containing bothreporter and quencher fluorescent dyes. When the probe is intact thereporter dye emission is negated due to the close proximity of thequencher dye. During PCR extension using additional gene-specificforward and reverse primers, the probe is cleaved by 5′ nucleaseactivity of Taq polymerase which releases the reporter dye from theprobe resulting in an increase in fluorescent emission.

[0355] The primers and probes used for real-time quantitative RT-PCRanalyses of Zcytor17 expression were designed using the primer designsoftware Primer Express™ (PE Applied Biosystems, Foster City, Calif.).Primers for human Zcytor17 were designed spanning an intron-exonjunction to eliminate amplification of genomic DNA. The forward primer,ZC37,877 (SEQ ID NO:81) and the reverse primer, ZC37,876 (SEQ ID NO:82)were used in a PCR reaction (below) at about 300 nM concentration tosynthesize a 73 bp product. The corresponding Zcytor17 TaqMan® probe,designated ZG37,776 (SEQ ID NO:83) was synthesized and labeled by PEApplied Biosystems. The ZG37,776 probe was labeled at the 5′end with areporter fluorescent dye (6-carboxy-fluorescein) (FAM) (PE AppliedBiosystems) and at the 3′ end with a quencher fluorescent dye(6-carboxy-tetramethyl-rhodamine) (TAMRA) (PE Applied Biosystems).

[0356] As a control to test the integrity and quality of RNA samplestested, all RNA samples (below) were screened for rRNA using a primerand probe set ordered from PE Applied Biosystems (cat #4304483). The kitcontains the rRNA forward primer (SEQ ID NO:84), the rRNA reverse primer(SEQ ID NO:85), and the rRNA TaqMan® probe (SEQ ID NO:86) The rRNA probewas labeled at the 5′end with a reporter fluorescent dye VIC (PE AppliedBiosystems) and at the 3′ end with the quencher fluorescent dye TAMRA(PE Applied Biosystems). The rRNA results also serve as an endogenouscontrol and allow for the normalization of the Zcytor17 mRNA expressionresults seen in the test samples.

[0357] Blood was drawn from several anonymous donors and PBMC'sisolated. Various immune cell subsets (CD3+, CD4+, CD8+, CD14+, CD19+,CD45RA, CD45RO and CD56+) were then isolated using Microbeads and theMagnetic Cell Separation System from Miltenyi Biotec. RNA was preparedfrom all of the CD45RA, CD45RO and CD56+ populations in their restingstate using an RNeasy Midiprep™ Kit (Qiagen, Valencia, Calif.) as permanufacturer's instruction. The CD3+, CD4+, and CD8+ populations wereactivated using 200 ng/ml plate-bound anti-CD3 antibody and 5 ug/mlsoluble anti-CD28 antibody and cells were collected for RNA isolation at0, 4 and 16 hours. The CD19+ samples were isolated from human tonsil andactivated with 0.5 ug/ml ionomycin and 10 ng/ml PMA. Cells were thencollected at 0, 4 hours and 24 hours and RNA isolated. Human CD14+monocytes were activated with either 0.1 ug/ml LPS or 1.0 ug/ml LPS for20 hours. Resting and activated cells were then collected and RNAisolated. In addition, RNA was isolated from resting and activated (10.0ug/ml LPS) human monocyte cell lines HL-60, THP-1 (ATCC No. TIB-202) andU937. THP-1 RNA was used as a control because it was shown to expressZcytor17 by Northern Blot (Example 3).

[0358] B. Real-time Quantitative RT-PCR

[0359] Relative levels of Zcytor17 mRNA were determined by analyzingtotal RNA samples using the one-step RT-PCR method (PE AppliedBiosystems). Total RNA from Zcytor17 expressing THP-1 cells was isolatedby standard methods and used to generate a standard curve used forquantitation. The curve consisted of 10-fold serial dilutions rangingfrom 0.25-0.00025 ng/μl for the rRNA screen and 250-0.25 ng/μl for theZcytor17 screen with each standard curve point analyzed in triplicate.The total RNA samples from the human cells were also analyzed intriplicate for human Zcytor17 transcript levels and for levels of rRNAas an endogenous control. In a total volume of 25 μl, each RNA samplewas subjected to a One-Step RT-PCR reaction containing: approximately 50ng of total RNA in buffer A (50 mM KCL, 10 mM Tris-HCL); the internalstandard dye, carboxy-x-rhodamine (ROX)); appropriate primers(approximately 50 nM rRNA primers (SEQ ID NO:84 and SEQ ID NO:85) forthe rRNA samples; and approximately 300 nM ZC37,877 (SEQ ID NO:81) andZC22,276 (SEQ ID NO:87) primers for Zcytor17 samples); the appropriateprobe (approximately 50 nM rRNA TaqMan® probe (SEQ ID NO:86) for rRNAsamples, approximately 100 nM ZG37,776 (SEQ ID NO:83) probe for Zcytor17samples); 5.5 mM MgCl₂; 300 μM each d-CTP, d-ATP, and d-GTP and 600 μMof d-UTP; MuLV reverse transcriptase (0.25 U/μl); AmpliTaq™ Gold DNApolymerase (0.025 U/μl) (PE Applied Biosystems); and RNase Inhibitor(0.4 U/μl) (PE Applied Biosystems). PCR thermal cycling conditions wereas follows: an initial reverse transcription (RT) step of one cycle at48° C. for 30 minutes; followed by an AmpliTaq Gold™ (PE AppliedBiosystems) activation step of one cycle at 95° C. for 10 minutes;followed by 40 cycles of amplification at 95° C. for 15 seconds and 60°C. for 1 minute.

[0360] Relative Zcytor17 RNA levels were determined by using theStandard Curve Method as described by the manufacturer, PE Biosystems(User Bulletin #2: ABI Prism 7700 Sequence Detection System, RelativeQuantitation of Gene Expression, Dec. 11, 1997). The rRNA measurementswere used to normalize the Zcytor17 levels. Three experiments were donetesting the aforementioned. Data shown in Tables 8 and 9 below areexpressed as a ratio of Zcytor17 mRNA to rRNA. TABLE 8 4 hr 16 hr 24 hrSample Resting Stimulation Stimulation Stimulation CD19+ PBMC 0.06 CD19+Tonsil 0.003 0.02 .002 CD3+ 0 0.72 0.51 CD45RA 0 CD45RO 0 CD56+ NK 0

[0361] TABLE 9 4 hr 16 hr Sample Resting Stimulation Stimulation CD4+ TCell 0.003 0.55 0.41 CD8+ T Cell 0.00 0.37 0.13

[0362] While there was some expression of Zcytor17 message in restingCD19+ B cells from the peripheral blood, both resting and activatedCD19+ B cells isolated from human tonsil showed only minimal expression.Resting memory T cells (CD45RO), naive T Cells (CD45RA) and NK cells(CD56+) all tested negative for Zcytor17 message. However, in CD3+ Tcells, Zcytor17 message underwent a dramatic upregulation to a ratio ofabout 0.72 following a 4 hour activation with anti-CD3 and anti-CD28antibodies. The ratio then dropped to 0.51 by 16 hours post-activation.Prior to activation, no Zcytor17 mRNA was detected in resting CD3+ Tcells.

[0363] The results revealed that Zcytor17 was not present in appreciablelevels in resting CD4+ or CD8+ T cell subsets. Following a 4 houractivation with anti-CD3 and anti-CD28 antibodies there appears to be asubstantial upregulation of Zcytor17 message produced in both CD4+ andCD8+ T cell subsets. By 16 hours post-activation, the Zcytor17 mRNA haddecreased to 0.41 in CD4+ T cells and 0.13 in CD8+ T cells.

[0364] There was extremely high expression of Zcytor17 message in boththe resting and activated monocyte cell lines THP-1 and U937. Theactivated U937's have the highest level of expression. HL-60's, apre-differentiated monocyte cell line, showed a decrease in Zcytor17mRNA expression upon activation. These results support expressionresults done by northern blot (Example 2). There was very littleexpression of Zcytor17 message in primary CD14+ monocytes both restingand activated with 0.1 ug/ml and 1.0 ug/ml LPS.

[0365] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 93 1 2402 DNA Homo sapiens CDS (171)...(2366) 1 ggcacgaggt gtgtgtgcagtatgaaaatt gagacaggaa ggcagagtgt cagcttgttc 60 cacctcagct gggaatgtgcatcaggcaac tcaagttttt caccacggca tgtgtctgtg 120 aatgtccgca aaacattctctctccccagc cttcatgtgt taacctgggg atg atg 176 Met Met 1 tgg acc tgg gcactg tgg atg ctc ccc tca ctc tgc aaa ttc agc ctg 224 Trp Thr Trp Ala LeuTrp Met Leu Pro Ser Leu Cys Lys Phe Ser Leu 5 10 15 gca gct ctg cca gctaag cct gag aac att tcc tgt gtc tac tac tat 272 Ala Ala Leu Pro Ala LysPro Glu Asn Ile Ser Cys Val Tyr Tyr Tyr 20 25 30 agg aaa aat tta acc tgcact tgg agt cca gga aag gaa acc agt tat 320 Arg Lys Asn Leu Thr Cys ThrTrp Ser Pro Gly Lys Glu Thr Ser Tyr 35 40 45 50 acc cag tac aca gtt aagaga act tac gct ttt gga gaa aaa cat gat 368 Thr Gln Tyr Thr Val Lys ArgThr Tyr Ala Phe Gly Glu Lys His Asp 55 60 65 aat tgt aca acc aat agt tctaca agt gaa aat cgt gct tcg tgc tct 416 Asn Cys Thr Thr Asn Ser Ser ThrSer Glu Asn Arg Ala Ser Cys Ser 70 75 80 ttt ttc ctt cca aga ata acg atccca gat aat tat acc att gag gtg 464 Phe Phe Leu Pro Arg Ile Thr Ile ProAsp Asn Tyr Thr Ile Glu Val 85 90 95 gaa gct gaa aat gga gat ggt gta attaaa tct cat atg aca tac tgg 512 Glu Ala Glu Asn Gly Asp Gly Val Ile LysSer His Met Thr Tyr Trp 100 105 110 aga tta gag aac ata gcg aaa act gaacca cct aag att ttc cgt gtg 560 Arg Leu Glu Asn Ile Ala Lys Thr Glu ProPro Lys Ile Phe Arg Val 115 120 125 130 aaa cca gtt ttg ggc atc aaa cgaatg att caa att gaa tgg ata aag 608 Lys Pro Val Leu Gly Ile Lys Arg MetIle Gln Ile Glu Trp Ile Lys 135 140 145 cct gag ttg gcg cct gtt tca tctgat tta aaa tac aca ctt cga ttc 656 Pro Glu Leu Ala Pro Val Ser Ser AspLeu Lys Tyr Thr Leu Arg Phe 150 155 160 agg aca gtc aac agt acc agc tggatg gaa gtc aac ttc gct aag aac 704 Arg Thr Val Asn Ser Thr Ser Trp MetGlu Val Asn Phe Ala Lys Asn 165 170 175 cgt aag gat aaa aac caa acg tacaac ctc acg ggg ctg cag cct ttt 752 Arg Lys Asp Lys Asn Gln Thr Tyr AsnLeu Thr Gly Leu Gln Pro Phe 180 185 190 aca gaa tat gtc ata gct ctg cgatgt gcg gtc aag gag tca aag ttc 800 Thr Glu Tyr Val Ile Ala Leu Arg CysAla Val Lys Glu Ser Lys Phe 195 200 205 210 tgg agt gac tgg agc caa gaaaaa atg gga atg act gag gaa gaa gct 848 Trp Ser Asp Trp Ser Gln Glu LysMet Gly Met Thr Glu Glu Glu Ala 215 220 225 cca tgt ggc ctg gaa ctg tggaga gtc ctg aaa cca gct gag gcg gat 896 Pro Cys Gly Leu Glu Leu Trp ArgVal Leu Lys Pro Ala Glu Ala Asp 230 235 240 gga aga agg cca gtg cgg ttgtta tgg aag aag gca aga gga gcc cca 944 Gly Arg Arg Pro Val Arg Leu LeuTrp Lys Lys Ala Arg Gly Ala Pro 245 250 255 gtc cta gag aaa aca ctt ggctac aac ata tgg tac tat cca gaa agc 992 Val Leu Glu Lys Thr Leu Gly TyrAsn Ile Trp Tyr Tyr Pro Glu Ser 260 265 270 aac act aac ctc aca gaa acaatg aac act act aac cag cag ctt gaa 1040 Asn Thr Asn Leu Thr Glu Thr MetAsn Thr Thr Asn Gln Gln Leu Glu 275 280 285 290 ctg cat ctg gga ggc gagagc ttt tgg gtg tct atg att tct tat aat 1088 Leu His Leu Gly Gly Glu SerPhe Trp Val Ser Met Ile Ser Tyr Asn 295 300 305 tct ctt ggg aag tct ccagtg gcc acc ctg agg att cca gct att caa 1136 Ser Leu Gly Lys Ser Pro ValAla Thr Leu Arg Ile Pro Ala Ile Gln 310 315 320 gaa aaa tca ttt cag tgcatt gag gtc atg cag gcc tgc gtt gct gag 1184 Glu Lys Ser Phe Gln Cys IleGlu Val Met Gln Ala Cys Val Ala Glu 325 330 335 gac cag cta gtg gtg aagtgg caa agc tct gct cta gac gtg aac act 1232 Asp Gln Leu Val Val Lys TrpGln Ser Ser Ala Leu Asp Val Asn Thr 340 345 350 tgg atg att gaa tgg tttccg gat gtg gac tca gag ccc acc acc ctt 1280 Trp Met Ile Glu Trp Phe ProAsp Val Asp Ser Glu Pro Thr Thr Leu 355 360 365 370 tcc tgg gaa tct gtgtct cag gcc acg aac tgg acg atc cag caa gat 1328 Ser Trp Glu Ser Val SerGln Ala Thr Asn Trp Thr Ile Gln Gln Asp 375 380 385 aaa tta aaa cct ttctgg tgc tat aac atc tct gtg tat cca atg ttg 1376 Lys Leu Lys Pro Phe TrpCys Tyr Asn Ile Ser Val Tyr Pro Met Leu 390 395 400 cat gac aaa gtt ggcgag cca tat tcc atc cag gct tat gcc aaa gaa 1424 His Asp Lys Val Gly GluPro Tyr Ser Ile Gln Ala Tyr Ala Lys Glu 405 410 415 ggc gtt cca tca gaaggt cct gag acc aag gtg gag aac att ggc gtg 1472 Gly Val Pro Ser Glu GlyPro Glu Thr Lys Val Glu Asn Ile Gly Val 420 425 430 aag acg gtc acg atcaca tgg aaa gag att ccc aag agt gag aga aag 1520 Lys Thr Val Thr Ile ThrTrp Lys Glu Ile Pro Lys Ser Glu Arg Lys 435 440 445 450 ggt atc atc tgcaac tac acc atc ttt tac caa gct gaa ggt gga aaa 1568 Gly Ile Ile Cys AsnTyr Thr Ile Phe Tyr Gln Ala Glu Gly Gly Lys 455 460 465 gga ttc tcc aagaca gtc aat tcc agc atc ttg cag tac ggc ctg gag 1616 Gly Phe Ser Lys ThrVal Asn Ser Ser Ile Leu Gln Tyr Gly Leu Glu 470 475 480 tcc ctg aaa cgaaag acc tct tac att gtt cag gtc atg gcc agc acc 1664 Ser Leu Lys Arg LysThr Ser Tyr Ile Val Gln Val Met Ala Ser Thr 485 490 495 agt gct ggg ggaacc aac ggg acc agc ata aat ttc aag aca ttg tca 1712 Ser Ala Gly Gly ThrAsn Gly Thr Ser Ile Asn Phe Lys Thr Leu Ser 500 505 510 ttc agt gtc tttgag att atc ctc ata act tct ctg att ggt gga ggc 1760 Phe Ser Val Phe GluIle Ile Leu Ile Thr Ser Leu Ile Gly Gly Gly 515 520 525 530 ctt ctt attctc att atc ctg aca gtg gca tat ggt ctc aaa aaa ccc 1808 Leu Leu Ile LeuIle Ile Leu Thr Val Ala Tyr Gly Leu Lys Lys Pro 535 540 545 aac aaa ttgact cat ctg tgt tgg ccc acc gtt ccc aac cct gct gaa 1856 Asn Lys Leu ThrHis Leu Cys Trp Pro Thr Val Pro Asn Pro Ala Glu 550 555 560 agt agt atagcc aca tgg cat gga gat gat ttc aag gat aag cta aac 1904 Ser Ser Ile AlaThr Trp His Gly Asp Asp Phe Lys Asp Lys Leu Asn 565 570 575 ctg aag gagtct gat gac tct gtg aac aca gaa gac agg atc tta aaa 1952 Leu Lys Glu SerAsp Asp Ser Val Asn Thr Glu Asp Arg Ile Leu Lys 580 585 590 cca tgt tccacc ccc agt gac aag ttg gtg att gac aag ttg gtg gtg 2000 Pro Cys Ser ThrPro Ser Asp Lys Leu Val Ile Asp Lys Leu Val Val 595 600 605 610 aac tttggg aat gtt ctg caa gaa att ttc aca gat gaa gcc aga acg 2048 Asn Phe GlyAsn Val Leu Gln Glu Ile Phe Thr Asp Glu Ala Arg Thr 615 620 625 ggt caggaa aac aat tta gga ggg gaa aag aat ggg tat gtg acc tgc 2096 Gly Gln GluAsn Asn Leu Gly Gly Glu Lys Asn Gly Tyr Val Thr Cys 630 635 640 ccc ttcagg cct gat tgt ccc ctg ggg aaa agt ttt gag gag ctc cca 2144 Pro Phe ArgPro Asp Cys Pro Leu Gly Lys Ser Phe Glu Glu Leu Pro 645 650 655 gtt tcacct gag att ccg ccc aga aaa tcc caa tac cta cgt tcg agg 2192 Val Ser ProGlu Ile Pro Pro Arg Lys Ser Gln Tyr Leu Arg Ser Arg 660 665 670 atg ccagag ggg acc cgc cca gaa gcc aaa gag cag ctt ctc ttt tct 2240 Met Pro GluGly Thr Arg Pro Glu Ala Lys Glu Gln Leu Leu Phe Ser 675 680 685 690 ggtcaa agt tta gta cca gat cat ctg tgt gag gaa gga gcc cca aat 2288 Gly GlnSer Leu Val Pro Asp His Leu Cys Glu Glu Gly Ala Pro Asn 695 700 705 ccatat ttg aaa aat tca gtg aca gcc agg gaa ttt ctt gtg tct gaa 2336 Pro TyrLeu Lys Asn Ser Val Thr Ala Arg Glu Phe Leu Val Ser Glu 710 715 720 aaactt cca gag cac acc aag gga gaa gtc taaatgcgac catagcatga 2386 Lys LeuPro Glu His Thr Lys Gly Glu Val 725 730 gaccctcggg gcctca 2402 2 732 PRTHomo sapiens 2 Met Met Trp Thr Trp Ala Leu Trp Met Leu Pro Ser Leu CysLys Phe 1 5 10 15 Ser Leu Ala Ala Leu Pro Ala Lys Pro Glu Asn Ile SerCys Val Tyr 20 25 30 Tyr Tyr Arg Lys Asn Leu Thr Cys Thr Trp Ser Pro GlyLys Glu Thr 35 40 45 Ser Tyr Thr Gln Tyr Thr Val Lys Arg Thr Tyr Ala PheGly Glu Lys 50 55 60 His Asp Asn Cys Thr Thr Asn Ser Ser Thr Ser Glu AsnArg Ala Ser 65 70 75 80 Cys Ser Phe Phe Leu Pro Arg Ile Thr Ile Pro AspAsn Tyr Thr Ile 85 90 95 Glu Val Glu Ala Glu Asn Gly Asp Gly Val Ile LysSer His Met Thr 100 105 110 Tyr Trp Arg Leu Glu Asn Ile Ala Lys Thr GluPro Pro Lys Ile Phe 115 120 125 Arg Val Lys Pro Val Leu Gly Ile Lys ArgMet Ile Gln Ile Glu Trp 130 135 140 Ile Lys Pro Glu Leu Ala Pro Val SerSer Asp Leu Lys Tyr Thr Leu 145 150 155 160 Arg Phe Arg Thr Val Asn SerThr Ser Trp Met Glu Val Asn Phe Ala 165 170 175 Lys Asn Arg Lys Asp LysAsn Gln Thr Tyr Asn Leu Thr Gly Leu Gln 180 185 190 Pro Phe Thr Glu TyrVal Ile Ala Leu Arg Cys Ala Val Lys Glu Ser 195 200 205 Lys Phe Trp SerAsp Trp Ser Gln Glu Lys Met Gly Met Thr Glu Glu 210 215 220 Glu Ala ProCys Gly Leu Glu Leu Trp Arg Val Leu Lys Pro Ala Glu 225 230 235 240 AlaAsp Gly Arg Arg Pro Val Arg Leu Leu Trp Lys Lys Ala Arg Gly 245 250 255Ala Pro Val Leu Glu Lys Thr Leu Gly Tyr Asn Ile Trp Tyr Tyr Pro 260 265270 Glu Ser Asn Thr Asn Leu Thr Glu Thr Met Asn Thr Thr Asn Gln Gln 275280 285 Leu Glu Leu His Leu Gly Gly Glu Ser Phe Trp Val Ser Met Ile Ser290 295 300 Tyr Asn Ser Leu Gly Lys Ser Pro Val Ala Thr Leu Arg Ile ProAla 305 310 315 320 Ile Gln Glu Lys Ser Phe Gln Cys Ile Glu Val Met GlnAla Cys Val 325 330 335 Ala Glu Asp Gln Leu Val Val Lys Trp Gln Ser SerAla Leu Asp Val 340 345 350 Asn Thr Trp Met Ile Glu Trp Phe Pro Asp ValAsp Ser Glu Pro Thr 355 360 365 Thr Leu Ser Trp Glu Ser Val Ser Gln AlaThr Asn Trp Thr Ile Gln 370 375 380 Gln Asp Lys Leu Lys Pro Phe Trp CysTyr Asn Ile Ser Val Tyr Pro 385 390 395 400 Met Leu His Asp Lys Val GlyGlu Pro Tyr Ser Ile Gln Ala Tyr Ala 405 410 415 Lys Glu Gly Val Pro SerGlu Gly Pro Glu Thr Lys Val Glu Asn Ile 420 425 430 Gly Val Lys Thr ValThr Ile Thr Trp Lys Glu Ile Pro Lys Ser Glu 435 440 445 Arg Lys Gly IleIle Cys Asn Tyr Thr Ile Phe Tyr Gln Ala Glu Gly 450 455 460 Gly Lys GlyPhe Ser Lys Thr Val Asn Ser Ser Ile Leu Gln Tyr Gly 465 470 475 480 LeuGlu Ser Leu Lys Arg Lys Thr Ser Tyr Ile Val Gln Val Met Ala 485 490 495Ser Thr Ser Ala Gly Gly Thr Asn Gly Thr Ser Ile Asn Phe Lys Thr 500 505510 Leu Ser Phe Ser Val Phe Glu Ile Ile Leu Ile Thr Ser Leu Ile Gly 515520 525 Gly Gly Leu Leu Ile Leu Ile Ile Leu Thr Val Ala Tyr Gly Leu Lys530 535 540 Lys Pro Asn Lys Leu Thr His Leu Cys Trp Pro Thr Val Pro AsnPro 545 550 555 560 Ala Glu Ser Ser Ile Ala Thr Trp His Gly Asp Asp PheLys Asp Lys 565 570 575 Leu Asn Leu Lys Glu Ser Asp Asp Ser Val Asn ThrGlu Asp Arg Ile 580 585 590 Leu Lys Pro Cys Ser Thr Pro Ser Asp Lys LeuVal Ile Asp Lys Leu 595 600 605 Val Val Asn Phe Gly Asn Val Leu Gln GluIle Phe Thr Asp Glu Ala 610 615 620 Arg Thr Gly Gln Glu Asn Asn Leu GlyGly Glu Lys Asn Gly Tyr Val 625 630 635 640 Thr Cys Pro Phe Arg Pro AspCys Pro Leu Gly Lys Ser Phe Glu Glu 645 650 655 Leu Pro Val Ser Pro GluIle Pro Pro Arg Lys Ser Gln Tyr Leu Arg 660 665 670 Ser Arg Met Pro GluGly Thr Arg Pro Glu Ala Lys Glu Gln Leu Leu 675 680 685 Phe Ser Gly GlnSer Leu Val Pro Asp His Leu Cys Glu Glu Gly Ala 690 695 700 Pro Asn ProTyr Leu Lys Asn Ser Val Thr Ala Arg Glu Phe Leu Val 705 710 715 720 SerGlu Lys Leu Pro Glu His Thr Lys Gly Glu Val 725 730 3 5 PRT ArtificialSequence WSXWS peptide motif 3 Trp Ser Xaa Trp Ser 1 5 4 2196 DNAArtificial Sequence Degenerate polynucleotide sequence of SEQ ID NO2 4atgatgtgga cntgggcnyt ntggatgytn ccnwsnytnt gyaarttyws nytngcngcn 60ytnccngcna arccngaraa yathwsntgy gtntaytayt aymgnaaraa yytnacntgy 120acntggwsnc cnggnaarga racnwsntay acncartaya cngtnaarmg nacntaygcn 180ttyggngara arcaygayaa ytgyacnacn aaywsnwsna cnwsngaraa ymgngcnwsn 240tgywsnttyt tyytnccnmg nathacnath ccngayaayt ayacnathga rgtngargcn 300garaayggng ayggngtnat haarwsncay atgacntayt ggmgnytnga raayathgcn 360aaracngarc cnccnaarat httymgngtn aarccngtny tnggnathaa rmgnatgath 420carathgart ggathaarcc ngarytngcn ccngtnwsnw sngayytnaa rtayacnytn 480mgnttymgna cngtnaayws nacnwsntgg atggargtna ayttygcnaa raaymgnaar 540gayaaraayc aracntayaa yytnacnggn ytncarccnt tyacngarta ygtnathgcn 600ytnmgntgyg cngtnaarga rwsnaartty tggwsngayt ggwsncarga raaratgggn 660atgacngarg argargcncc ntgyggnytn garytntggm gngtnytnaa rccngcngar 720gcngayggnm gnmgnccngt nmgnytnytn tggaaraarg cnmgnggngc nccngtnytn 780garaaracny tnggntayaa yathtggtay tayccngarw snaayacnaa yytnacngar 840acnatgaaya cnacnaayca rcarytngar ytncayytng gnggngarws nttytgggtn 900wsnatgathw sntayaayws nytnggnaar wsnccngtng cnacnytnmg nathccngcn 960athcargara arwsnttyca rtgyathgar gtnatgcarg cntgygtngc ngargaycar 1020ytngtngtna artggcarws nwsngcnytn gaygtnaaya cntggatgat hgartggtty 1080ccngaygtng aywsngarcc nacnacnytn wsntgggarw sngtnwsnca rgcnacnaay 1140tggacnathc arcargayaa rytnaarccn ttytggtgyt ayaayathws ngtntayccn 1200atgytncayg ayaargtngg ngarccntay wsnathcarg cntaygcnaa rgarggngtn 1260ccnwsngarg gnccngarac naargtngar aayathggng tnaaracngt nacnathacn 1320tggaargara thccnaarws ngarmgnaar ggnathatht gyaaytayac nathttytay 1380cargcngarg gnggnaargg nttywsnaar acngtnaayw snwsnathyt ncartayggn 1440ytngarwsny tnaarmgnaa racnwsntay athgtncarg tnatggcnws nacnwsngcn 1500ggnggnacna ayggnacnws nathaaytty aaracnytnw snttywsngt nttygarath 1560athytnatha cnwsnytnat hggnggnggn ytnytnathy tnathathyt nacngtngcn 1620tayggnytna araarccnaa yaarytnacn cayytntgyt ggccnacngt nccnaayccn 1680gcngarwsnw snathgcnac ntggcayggn gaygayttya argayaaryt naayytnaar 1740garwsngayg aywsngtnaa yacngargay mgnathytna arccntgyws nacnccnwsn 1800gayaarytng tnathgayaa rytngtngtn aayttyggna aygtnytnca rgarathtty 1860acngaygarg cnmgnacngg ncargaraay aayytnggng gngaraaraa yggntaygtn 1920acntgyccnt tymgnccnga ytgyccnytn ggnaarwsnt tygargaryt nccngtnwsn 1980ccngarathc cnccnmgnaa rwsncartay ytnmgnwsnm gnatgccnga rggnacnmgn 2040ccngargcna argarcaryt nytnttywsn ggncarwsny tngtnccnga ycayytntgy 2100gargarggng cnccnaaycc ntayytnaar aaywsngtna cngcnmgnga rttyytngtn 2160wsngaraary tnccngarca yacnaarggn gargtn 2196 5 20 DNA ArtificialSequence Oligonucleotide primer ZC12701 5 tcagaggtaa ctcccgttgc 20 6 23DNA Artificial Sequence Oligonucleotide primer ZC27898 6 ttagcgcagagcagccatac acc 23 7 25 DNA Artificial Sequence Oligonucleotide primerZC14063 7 caccagacat aatagctgac agact 25 8 23 DNA Artificial SequenceOligonucleotide primer ZC27899 8 ccagaacttt gactccttga ccg 23 9 765 DNAHomo sapiens 9 tgtgtgtgca gtatgaaaat tgagacagga aggcagagtg tcagcttgttccacctcagc 60 tgggaatgtg catcaggcaa ctcaagtttt tcaccacggc atgtgtctgtgaatgtccgc 120 aaaacattct ctctccccag ccttcatgtg ttaacctggg gatgatgtggacctgggcac 180 tgtggatgct cccttcactc tgcaaattca gcctggcagc tctgccagctaagcctgaga 240 acatttcctg tgtctactac tataggaaaa atttaacctg cacttggagtccaggaaagg 300 aaaccagtta tacccagtac acagttaaga gaacttacgc ttttggagaaaaacatgata 360 attgtacaac caatagttct acaagtgaaa atcgtgcttc gtgctcttttttccttccaa 420 gaataacgat cccagataat tataccattg aggtggaagc tgaaaatggagatggtgtaa 480 ttaaatctca tatgacatac tggagattag agaacatagc gaaaactgaaccacctaaga 540 ttttccgtgt gaaaccagtt ttgggcatca aacgaatgat tcaaattgaatggataaagc 600 ctgagttggc gcctgtttca tctgatttaa aatacacact tcgattcaggacagtcaaca 660 gtaccagctg gatggaagtc aacttcgcta agaaccgtaa ggataaaaaccaaacgtaca 720 acctcacggg gctgcagcct tttacagaat atgtcatagc tctgc 765 1024 DNA Artificial Sequence Oligonucleotide primer ZC28481 10 gaatggataaagcctgagtt ggcg 24 11 20 DNA Artificial Sequence Oligonucleotide primerZC6346 11 ggccccttgc tccataccac 20 12 24 DNA Artificial SequenceOligonucleotide primer ZC28480 12 cgattcagga cagtcaacag tacc 24 13 24DNA Artificial Sequence Oligonucleotide primer ZC26405 13 tatagaaggacacctagtca gaca 24 14 23 DNA Artificial Sequence Oligonucleotide primerZC27895 14 gaagtcaact tcgctaagaa ccg 23 15 21 DNA Artificial SequenceOligonucleotide primer ZC5020 15 cactggagtg gcaacttcca g 21 16 1853 DNAHomo sapiens 16 agtcaacttc gctaagaacc gtaaggataa aaaccaaacg tacaacctcacggggctgca 60 gccttttaca gaatatgtca tagctctgcg atgtgcggtc aaggagtcaaagttctggag 120 tgactggagc caagaaaaaa tgggaatgac tgaggaagaa gctccatgtggcctggaact 180 gtggggagtc ctgaaaccag ctgaggcgga tggaagaagg ccagtgcggttgttatggaa 240 gaaggcaaga ggagccccag tcctagagaa aacacttggc tacaacatatggtactatcc 300 agaaagcaac actaacctca cagaaacaat gaacactact aaccagcagcttgaactgca 360 tctgggaggc gagagctttt gggtgtctat gatttcttat aattctcttgggaagtctcc 420 agtggccacc ctgaggattc cagctattca agaaaaatca tttcagtgcattgaggtcat 480 gcaggcctgc gttgctgagg accagctagt ggtgaagtgg caaagctctgctctagacgt 540 gaacacttgg atgattgaat ggtttccgga tgtggactca gagcccaccaccctttcctg 600 ggaatctgtg tctcaggcca cgaactggac gatccagcaa gataaattaaaacctttctg 660 gtgctataac atctctgtgt atccaatgtt gcatgacaaa gttggcgagccatattccat 720 ccaggcttat gccaaagaag gcgttccatc agaaggtcct gagaccaaggtggagaacat 780 tggcgtgaag acggtcacga tcacatggag agagattccc aagagtgagagaaagggtat 840 catctgcaac tacaccatct tttaccaagc tgaaggtgga aaaggattctccaagacagt 900 caattccagc atcttgcagt acggcctgga gtccctgaaa cgaaagacctcttacattgt 960 tcaggtcatg gccagcacca gtgctggggg aaccaacggg accagcataaatttcaagac 1020 attgtcattc agtgtctttg agattatcct cataacttct ctgattggtggaggccttct 1080 tattctcatt atcctgacag tggcatatgg tctcaaaaaa cccaacaaattgactcatct 1140 gtgttggccc accgttccca accctgctga gagtagtata gccacacggcatggagatga 1200 tttcaaggat aagctaaacc tgaaggagtc tgatgactct gtgaacacagaagacaggat 1260 cttaaaacca tgttccaccc ccagtgacaa gttggtgatt gacaagttggtggtgaactt 1320 tgggaatgtt ctgcaagaaa ttttcacaga tgaagccaga acgggtcaggaaaacaattt 1380 aggaggggaa aagaatggga ctagaattct gtcttcctgc ccaacttcaatataagtgtg 1440 gactaaaatg cgagaaaggt gtcctgtggt ctatgcaaat tagaaaggacatgcagagtt 1500 ttccaactag gaagactgaa tctgtggccc caagagaacc atctctgaagactgggtatg 1560 tggtcttttc cacacatgga ccacctacgg atgtaatctg taatgcatgtgcatgagaag 1620 tctgttatta agtagagtgt gaaaacatgg ttatggtaat aggaacagcttttaaaatgc 1680 ttttgcattt gggcctttca tacaaaaaag ccataatacc attttcatgtaatgctatac 1740 ttctatacta ttttcatgta atactatact tctatactat tttcatgtaatactatactt 1800 ctatactatt ttcatgtaat actatacttc tatattaaag ttttacccactca 1853 17 1299 DNA Homo sapiens CDS (162)...(1133) 17 tgtgtgtgcagtatgaaaat tgagacagga aggcagagtg tcagcttgtt ccacctcagc 60 tgggaatgtgcatcaggcaa ctcaagtttt tcaccacggc atgtgtctgt gaatgtccgc 120 aaaacattctctctccccag ccttcatgtg ttaacctggg g atg atg tgg acc tgg 176 Met Met TrpThr Trp 1 5 gca ctg tgg atg ctc ccc tca ctc tgc aaa ttc agc ctg gca gctctg 224 Ala Leu Trp Met Leu Pro Ser Leu Cys Lys Phe Ser Leu Ala Ala Leu10 15 20 cca gct aag cct gag aac att tcc tgt gtc tac tac tat agg aaa aat272 Pro Ala Lys Pro Glu Asn Ile Ser Cys Val Tyr Tyr Tyr Arg Lys Asn 2530 35 tta acc tgc act tgg agt cca gga aag gaa acc agt tat acc cag tac320 Leu Thr Cys Thr Trp Ser Pro Gly Lys Glu Thr Ser Tyr Thr Gln Tyr 4045 50 aca gtt aag aga act tac gct ttt gga gaa aaa cat gat aat tgt aca368 Thr Val Lys Arg Thr Tyr Ala Phe Gly Glu Lys His Asp Asn Cys Thr 5560 65 acc aat agt tct aca agt gaa aat cgt gct tcg tgc tct ttt ttc ctt416 Thr Asn Ser Ser Thr Ser Glu Asn Arg Ala Ser Cys Ser Phe Phe Leu 7075 80 85 cca aga ata acg atc cca gat aat tat acc att gag gtg gaa gct gaa464 Pro Arg Ile Thr Ile Pro Asp Asn Tyr Thr Ile Glu Val Glu Ala Glu 9095 100 aat gga gat ggt gta att aaa tct cat atg aca tac tgg aga tta gag512 Asn Gly Asp Gly Val Ile Lys Ser His Met Thr Tyr Trp Arg Leu Glu 105110 115 aac ata gcg aaa act gaa cca cct aag att ttc cgt gtg aaa cca gtt560 Asn Ile Ala Lys Thr Glu Pro Pro Lys Ile Phe Arg Val Lys Pro Val 120125 130 ttg ggc atc aaa cga atg att caa att gaa tgg ata aag cct gag ttg608 Leu Gly Ile Lys Arg Met Ile Gln Ile Glu Trp Ile Lys Pro Glu Leu 135140 145 gcg cct gtt tca tct gat tta aaa tac aca ctt cga ttc agg aca gtc656 Ala Pro Val Ser Ser Asp Leu Lys Tyr Thr Leu Arg Phe Arg Thr Val 150155 160 165 aac agt acc agc tgg atg gaa gtc aac ttc gct aag aac cgt aaggat 704 Asn Ser Thr Ser Trp Met Glu Val Asn Phe Ala Lys Asn Arg Lys Asp170 175 180 aaa aac caa acg tac aac ctc acg ggg ctg cag cct ttt aca gaatat 752 Lys Asn Gln Thr Tyr Asn Leu Thr Gly Leu Gln Pro Phe Thr Glu Tyr185 190 195 gtc ata gct ctg cga tgt gcg gtc aag gag tca aag ttc tgg agtgac 800 Val Ile Ala Leu Arg Cys Ala Val Lys Glu Ser Lys Phe Trp Ser Asp200 205 210 tgg agc caa gaa aaa atg gga atg act gag gaa gaa gct cca tgtggc 848 Trp Ser Gln Glu Lys Met Gly Met Thr Glu Glu Glu Ala Pro Cys Gly215 220 225 ctg gaa ctg tgg aga gtc ctg aaa cca gct gag gcg gat gga agaagg 896 Leu Glu Leu Trp Arg Val Leu Lys Pro Ala Glu Ala Asp Gly Arg Arg230 235 240 245 cca gtg cgg ttg tta tgg aag aag gca aga gga gcc cca gtccta gag 944 Pro Val Arg Leu Leu Trp Lys Lys Ala Arg Gly Ala Pro Val LeuGlu 250 255 260 aaa aca ctt ggc tac aac ata tgg tac tat cca gaa agc aacact aac 992 Lys Thr Leu Gly Tyr Asn Ile Trp Tyr Tyr Pro Glu Ser Asn ThrAsn 265 270 275 ctc aca gaa aca atg aac act act aac cag cag ctt gaa ctgcat ctg 1040 Leu Thr Glu Thr Met Asn Thr Thr Asn Gln Gln Leu Glu Leu HisLeu 280 285 290 gga ggc gag agc ttt tgg gtg tct atg att tct tat aat tctctt ggg 1088 Gly Gly Glu Ser Phe Trp Val Ser Met Ile Ser Tyr Asn Ser LeuGly 295 300 305 aag tct cca gtg gcc acc ctg agg att cca gct att caa gaaaaa 1133 Lys Ser Pro Val Ala Thr Leu Arg Ile Pro Ala Ile Gln Glu Lys 310315 320 tagaaacttt acagatgcta gtcccagaca taaaagaaaa taatgttctggatgtgcacg 1193 atggctcacg cctgtaatcc cagcactttg aggccaagac gggtggatcgctgagttcag 1253 gagttcaaga caagtccagg caacatagtg aaaccttgtt tctaca 129918 324 PRT Homo sapiens 18 Met Met Trp Thr Trp Ala Leu Trp Met Leu ProSer Leu Cys Lys Phe 1 5 10 15 Ser Leu Ala Ala Leu Pro Ala Lys Pro GluAsn Ile Ser Cys Val Tyr 20 25 30 Tyr Tyr Arg Lys Asn Leu Thr Cys Thr TrpSer Pro Gly Lys Glu Thr 35 40 45 Ser Tyr Thr Gln Tyr Thr Val Lys Arg ThrTyr Ala Phe Gly Glu Lys 50 55 60 His Asp Asn Cys Thr Thr Asn Ser Ser ThrSer Glu Asn Arg Ala Ser 65 70 75 80 Cys Ser Phe Phe Leu Pro Arg Ile ThrIle Pro Asp Asn Tyr Thr Ile 85 90 95 Glu Val Glu Ala Glu Asn Gly Asp GlyVal Ile Lys Ser His Met Thr 100 105 110 Tyr Trp Arg Leu Glu Asn Ile AlaLys Thr Glu Pro Pro Lys Ile Phe 115 120 125 Arg Val Lys Pro Val Leu GlyIle Lys Arg Met Ile Gln Ile Glu Trp 130 135 140 Ile Lys Pro Glu Leu AlaPro Val Ser Ser Asp Leu Lys Tyr Thr Leu 145 150 155 160 Arg Phe Arg ThrVal Asn Ser Thr Ser Trp Met Glu Val Asn Phe Ala 165 170 175 Lys Asn ArgLys Asp Lys Asn Gln Thr Tyr Asn Leu Thr Gly Leu Gln 180 185 190 Pro PheThr Glu Tyr Val Ile Ala Leu Arg Cys Ala Val Lys Glu Ser 195 200 205 LysPhe Trp Ser Asp Trp Ser Gln Glu Lys Met Gly Met Thr Glu Glu 210 215 220Glu Ala Pro Cys Gly Leu Glu Leu Trp Arg Val Leu Lys Pro Ala Glu 225 230235 240 Ala Asp Gly Arg Arg Pro Val Arg Leu Leu Trp Lys Lys Ala Arg Gly245 250 255 Ala Pro Val Leu Glu Lys Thr Leu Gly Tyr Asn Ile Trp Tyr TyrPro 260 265 270 Glu Ser Asn Thr Asn Leu Thr Glu Thr Met Asn Thr Thr AsnGln Gln 275 280 285 Leu Glu Leu His Leu Gly Gly Glu Ser Phe Trp Val SerMet Ile Ser 290 295 300 Tyr Asn Ser Leu Gly Lys Ser Pro Val Ala Thr LeuArg Ile Pro Ala 305 310 315 320 Ile Gln Glu Lys 19 23 DNA ArtificialSequence Oligonucleotide primer ZC27897 19 caagctactt ctctggtgta tgg 2320 22 DNA Artificial Sequence Oligonucleotide primer ZC28521 20gagtagtagc tccaggattc ac 22 21 1476 DNA Homo sapiens CDS (162)...(878)21 tgtgtgtgca gtatgaaaat tgagacagga aggcagagtg tcagcttgtt ccacctcagc 60tgggaatgtg catcaggcaa ctcaagtttt tcaccacggc atgtgtctgt gaatgtccgc 120aaaacattct ctctccccag ccttcatgtg ttaacctggg g atg atg tgg acc tgg 176Met Met Trp Thr Trp 1 5 gca ctg tgg atg ctc ccc tca ctc tgc aaa ttc agcctg gca gct ctg 224 Ala Leu Trp Met Leu Pro Ser Leu Cys Lys Phe Ser LeuAla Ala Leu 10 15 20 cca gct aag cct gag aac att tcc tgt gtc tac tac tatagg aaa aat 272 Pro Ala Lys Pro Glu Asn Ile Ser Cys Val Tyr Tyr Tyr ArgLys Asn 25 30 35 tta acc tgc act tgg agt cca gga aag gaa acc agt tat acccag tac 320 Leu Thr Cys Thr Trp Ser Pro Gly Lys Glu Thr Ser Tyr Thr GlnTyr 40 45 50 aca gtt aag aga act tac gct ttt gga gaa aaa cat gat aat tgtaca 368 Thr Val Lys Arg Thr Tyr Ala Phe Gly Glu Lys His Asp Asn Cys Thr55 60 65 acc aat agt tct aca agt gaa aat cgt gct tcg tgc tct ttt ttc ctt416 Thr Asn Ser Ser Thr Ser Glu Asn Arg Ala Ser Cys Ser Phe Phe Leu 7075 80 85 cca aga ata acg atc cca gat aat tat acc att gag gtg gaa gct gaa464 Pro Arg Ile Thr Ile Pro Asp Asn Tyr Thr Ile Glu Val Glu Ala Glu 9095 100 aat gga gat ggt gta att aaa tct cat atg aca tac tgg aga tta gag512 Asn Gly Asp Gly Val Ile Lys Ser His Met Thr Tyr Trp Arg Leu Glu 105110 115 aac ata gcg aaa act gaa cca cct aag att ttc cgt gtg aaa cca gtt560 Asn Ile Ala Lys Thr Glu Pro Pro Lys Ile Phe Arg Val Lys Pro Val 120125 130 ttg ggc atc aaa cga atg att caa att gaa tgg ata aag cct gag ttg608 Leu Gly Ile Lys Arg Met Ile Gln Ile Glu Trp Ile Lys Pro Glu Leu 135140 145 gcg cct gtt tca tct gat tta aaa tac aca ctt cga ttc agg aca gtc656 Ala Pro Val Ser Ser Asp Leu Lys Tyr Thr Leu Arg Phe Arg Thr Val 150155 160 165 aac agt acc agc tgg atg gaa gtc aac ttc gct aag aac cgt aaggat 704 Asn Ser Thr Ser Trp Met Glu Val Asn Phe Ala Lys Asn Arg Lys Asp170 175 180 aaa aac caa acg tac aac ctc acg ggg ctg cag cct ttt aca gaatat 752 Lys Asn Gln Thr Tyr Asn Leu Thr Gly Leu Gln Pro Phe Thr Glu Tyr185 190 195 gtc ata gct ctg cga tgt gcg gtc aag gag tca aag ttc tgg agtgac 800 Val Ile Ala Leu Arg Cys Ala Val Lys Glu Ser Lys Phe Trp Ser Asp200 205 210 tgg agc caa gaa aaa atg gga atg act gag gaa gaa ggc aag ctactc 848 Trp Ser Gln Glu Lys Met Gly Met Thr Glu Glu Glu Gly Lys Leu Leu215 220 225 cct gcg att ccc gtc ctg tct gct ctg gtg tagggctgctttgggctaga 898 Pro Ala Ile Pro Val Leu Ser Ala Leu Val 230 235cttggtgggg tttgtcacca cctggttggg aatcatggaa tctcatgacc ccaggggccc 958cctgtaccat cgagagtgag cctgcacaac tttgtgcccc aaaggcaaag gatcacattt 1018taatactcat gaggttctta tactatacat gaaagggtat catatcattt gttttgtttt 1078gttttgtttt tgagatggag tcttactctg tcacccagga tggagtgcag tgatgtgatc 1138tcggctcact gccaccacca cctcccgagt tcaagcaatt cttgtgcctc agcctcccaa 1198gtagctggga ttacaggggc ccacgaccat gcccggttga tttttgtatt tttagtagag 1258aagggatatc accatgttgg ctaggctagt cttgaactcc tgacctcagg taatctgccc 1318accttgacct cccaaagtgt tgggattaca ggcgtgagcc actgtgcccc gccagtatca 1378tatcatctga aggtatcctg tgataaatta aagatacata ttgtgaatcc tggagctact 1438actcaaaaaa taaataaagg tgtaactaat acaattta 1476 22 239 PRT Homo sapiens22 Met Met Trp Thr Trp Ala Leu Trp Met Leu Pro Ser Leu Cys Lys Phe 1 510 15 Ser Leu Ala Ala Leu Pro Ala Lys Pro Glu Asn Ile Ser Cys Val Tyr 2025 30 Tyr Tyr Arg Lys Asn Leu Thr Cys Thr Trp Ser Pro Gly Lys Glu Thr 3540 45 Ser Tyr Thr Gln Tyr Thr Val Lys Arg Thr Tyr Ala Phe Gly Glu Lys 5055 60 His Asp Asn Cys Thr Thr Asn Ser Ser Thr Ser Glu Asn Arg Ala Ser 6570 75 80 Cys Ser Phe Phe Leu Pro Arg Ile Thr Ile Pro Asp Asn Tyr Thr Ile85 90 95 Glu Val Glu Ala Glu Asn Gly Asp Gly Val Ile Lys Ser His Met Thr100 105 110 Tyr Trp Arg Leu Glu Asn Ile Ala Lys Thr Glu Pro Pro Lys IlePhe 115 120 125 Arg Val Lys Pro Val Leu Gly Ile Lys Arg Met Ile Gln IleGlu Trp 130 135 140 Ile Lys Pro Glu Leu Ala Pro Val Ser Ser Asp Leu LysTyr Thr Leu 145 150 155 160 Arg Phe Arg Thr Val Asn Ser Thr Ser Trp MetGlu Val Asn Phe Ala 165 170 175 Lys Asn Arg Lys Asp Lys Asn Gln Thr TyrAsn Leu Thr Gly Leu Gln 180 185 190 Pro Phe Thr Glu Tyr Val Ile Ala LeuArg Cys Ala Val Lys Glu Ser 195 200 205 Lys Phe Trp Ser Asp Trp Ser GlnGlu Lys Met Gly Met Thr Glu Glu 210 215 220 Glu Gly Lys Leu Leu Pro AlaIle Pro Val Leu Ser Ala Leu Val 225 230 235 23 24 DNA ArtificialSequence Oligonucleotide primer ZC28575 23 ccaggaaagg aaaccagtta tacc 2424 23 DNA Artificial Sequence Oligonucleotide primer ZC27899 24ccagaacttt gactccttga ccg 23 25 25 DNA Artificial SequenceOligonucleotide primer ZC14063 25 caccagacat aatagctgac agact 25 26 21DNA Artificial Sequence Oligonucleotide primer ZC17574 26 ggtrttgctcagcatgcaca c 21 27 24 DNA Artificial Sequence Oligonucleotide primerZC17600 27 catgtaggcc atgaggtcca ccac 24 28 25 DNA Artificial SequenceOligonucleotide primer ZC26358 28 aaaaccaaac gtacaacctc acggg 25 29 25DNA Artificial Sequence Oligonucleotide primer ZC26359 29 gagcagccatacaccagagc agaca 25 30 33 DNA Artificial Sequence Oligonucleotide primerZC17212 30 ggggaattcg aagccatgcc ctcttgggcc ctc 33 31 30 DNA ArtificialSequence Oligonucleotide primer ZC17313 31 caccctgcga agccttagcagcagtaggcc 30 32 30 DNA Artificial Sequence Oligonucleotide primerZC17205 32 cccgccccat ccccgtggat caccttggtg 30 33 30 DNA ArtificialSequence Oligonucleotide primer ZC17206 33 gggtctagac cttcagggctgctgccaata 30 34 6 PRT Artificial Sequence Glu-Glu Tag peptide 34 GluTyr Met Pro Met Glu 1 5 35 8 PRT Artificial Sequence FLAG tag peptidesequence 35 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 36 699 DNA Homo sapiens36 gagcccagat cttcagacaa aactcacaca tgcccaccgt gcccagcacc tgaagccgag 60ggggcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc catcctccat cgagaaaacc 360atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660tacacgcaga agagcctctc cctgtctccg ggtaaataa 699 37 990 DNA Homo sapiensCDS (1)...(990) 37 gct agc acc aag ggc cca tcg gtc ttc ccc ctg gca ccctcc tcc aag 48 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro SerSer Lys 1 5 10 15 agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtcaag gac tac 96 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val LysAsp Tyr 20 25 30 ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctgacc agc 144 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu ThrSer 35 40 45 ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tactcc 192 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser50 55 60 ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc240 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 6570 75 80 tac atc tgc aac gtg aat cac aag ccc agc aac acc aag gtg gac aag288 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 8590 95 aaa gtt gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg tgc336 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100105 110 cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca384 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115120 125 aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc432 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130135 140 gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg480 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145150 155 160 tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgggag 528 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu165 170 175 gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtcctg 576 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu180 185 190 cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tccaac 624 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn195 200 205 aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaaggg 672 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly210 215 220 cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gatgag 720 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240 ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggcttc tat 768 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly PheTyr 245 250 255 ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccggag aac 816 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro GluAsn 260 265 270 aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tccttc ttc 864 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser PhePhe 275 280 285 ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag cagggg aac 912 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln GlyAsn 290 295 300 gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cactac acg 960 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His TyrThr 305 310 315 320 cag aag agc ctc tcc ctg tct ccg ggt aaa 990 Gln LysSer Leu Ser Leu Ser Pro Gly Lys 325 330 38 330 PRT Homo sapiens 38 AlaSer Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 7580 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 9095 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val ThrCys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys PheAsn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys ThrLys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val SerVal Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu TyrLys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu LysThr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val TyrThr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln ValSer Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile AlaVal Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys ThrThr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr SerLys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val PheSer Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 39 321 DNA Homo sapiensCDS (1)...(321) 39 act gtg gct gca cca tct gtc ttc atc ttc ccg cca tctgat gag cag 48 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser AspGlu Gln 1 5 10 15 ttg aaa tct ggt acc gcc tct gtt gtg tgc ctg ctg aataac ttc tat 96 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn AsnPhe Tyr 20 25 30 ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctccaa tcg 144 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu GlnSer 35 40 45 ggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac agcacc 192 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr50 55 60 tac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag aaa240 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 6570 75 80 cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc288 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 8590 95 gtc aca aag agc ttc aac agg gga gag tgt tag 321 Val Thr Lys SerPhe Asn Arg Gly Glu Cys * 100 105 40 106 PRT Homo sapiens 40 Thr Val AlaAla Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu LysSer Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro ArgGlu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly AsnSer Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr SerLeu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 HisLys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 ValThr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 41 8 DNA Artificial SequenceOligonucleotide primer ZC11440 41 aattgaga 8 42 8 DNA ArtificialSequence Oligonucleotide primer ZC11441 42 cgcgtctc 8 43 100 DNAArtificial Sequence Oligonucleotide primer ZC12749 43 gtaccttcccgtaaatccct ccccttcccg gaattacacc cgcgtatttc ccagaaaagg 60 aactgtagatttctaggaat tcaatccttg gccacgcgtc 100 44 100 DNA Artificial SequenceOligonucleotide primer ZC12748 44 tcgagacgcg tggccaagga ttgaattcctagaaatctac agttcctttt ctgggaaata 60 cgcgggtgta attccgggaa ggggagggatttacgggaag 100 45 2529 DNA Homo sapiens CDS (162)...(2108) 45 tgtgtgtgcagtatgaaaat tgagacagga aggcagagtg tcagcttgtt ccacctcagc 60 tgggaatgtgcatcaggcaa ctcaagtttt tcaccacggc atgtgtctgt gaatgtccgc 120 aaaacattctctctccccag ccttcatgtg ttaacctggg g atg atg tgg acc tgg 176 Met Met TrpThr Trp 1 5 gca ctg tgg atg ctc ccc tca ctc tgc aaa ttc agc ctg gca gctctg 224 Ala Leu Trp Met Leu Pro Ser Leu Cys Lys Phe Ser Leu Ala Ala Leu10 15 20 cca gct aag cct gag aac att tcc tgt gtc tac tac tat agg aaa aat272 Pro Ala Lys Pro Glu Asn Ile Ser Cys Val Tyr Tyr Tyr Arg Lys Asn 2530 35 tta acc tgc act tgg agt cca gga aag gaa acc agt tat acc cag tac320 Leu Thr Cys Thr Trp Ser Pro Gly Lys Glu Thr Ser Tyr Thr Gln Tyr 4045 50 aca gtt aag aga act tac gct ttt gga gaa aaa cat gat aat tgt aca368 Thr Val Lys Arg Thr Tyr Ala Phe Gly Glu Lys His Asp Asn Cys Thr 5560 65 acc aat agt tct aca agt gaa aat cgt gct tcg tgc tct ttt ttc ctt416 Thr Asn Ser Ser Thr Ser Glu Asn Arg Ala Ser Cys Ser Phe Phe Leu 7075 80 85 cca aga ata acg atc cca gat aat tat acc att gag gtg gaa gct gaa464 Pro Arg Ile Thr Ile Pro Asp Asn Tyr Thr Ile Glu Val Glu Ala Glu 9095 100 aat gga gat ggt gta att aaa tct cat atg aca tac tgg aga tta gag512 Asn Gly Asp Gly Val Ile Lys Ser His Met Thr Tyr Trp Arg Leu Glu 105110 115 aac ata gcg aaa act gaa cca cct aag att ttc cgt gtg aaa cca gtt560 Asn Ile Ala Lys Thr Glu Pro Pro Lys Ile Phe Arg Val Lys Pro Val 120125 130 ttg ggc atc aaa cga atg att caa att gaa tgg ata aag cct gag ttg608 Leu Gly Ile Lys Arg Met Ile Gln Ile Glu Trp Ile Lys Pro Glu Leu 135140 145 gcg cct gtt tca tct gat tta aaa tac aca ctt cga ttc agg aca gtc656 Ala Pro Val Ser Ser Asp Leu Lys Tyr Thr Leu Arg Phe Arg Thr Val 150155 160 165 aac agt acc agc tgg atg gaa gtc aac ttc gct aag aac cgt aaggat 704 Asn Ser Thr Ser Trp Met Glu Val Asn Phe Ala Lys Asn Arg Lys Asp170 175 180 aaa aac caa acg tac aac ctc acg ggg ctg cag cct ttt aca gaatat 752 Lys Asn Gln Thr Tyr Asn Leu Thr Gly Leu Gln Pro Phe Thr Glu Tyr185 190 195 gtc ata gct ctg cga tgt gcg gtc aag gag tca aag ttc tgg agtgac 800 Val Ile Ala Leu Arg Cys Ala Val Lys Glu Ser Lys Phe Trp Ser Asp200 205 210 tgg agc caa gaa aaa atg gga atg act gag gaa gaa gct cca tgtggc 848 Trp Ser Gln Glu Lys Met Gly Met Thr Glu Glu Glu Ala Pro Cys Gly215 220 225 ctg gaa ctg tgg aga gtc ctg aaa cca gct gag gcg gat gga agaagg 896 Leu Glu Leu Trp Arg Val Leu Lys Pro Ala Glu Ala Asp Gly Arg Arg230 235 240 245 cca gtg cgg ttg tta tgg aag aag gca aga gga gcc cca gtccta gag 944 Pro Val Arg Leu Leu Trp Lys Lys Ala Arg Gly Ala Pro Val LeuGlu 250 255 260 aaa aca ctt ggc tac aac ata tgg tac tat cca gaa agc aacact aac 992 Lys Thr Leu Gly Tyr Asn Ile Trp Tyr Tyr Pro Glu Ser Asn ThrAsn 265 270 275 ctc aca gaa aca atg aac act act aac cag cag ctt gaa ctgcat ctg 1040 Leu Thr Glu Thr Met Asn Thr Thr Asn Gln Gln Leu Glu Leu HisLeu 280 285 290 gga ggc gag agc ttt tgg gtg tct atg att tct tat aat tctctt ggg 1088 Gly Gly Glu Ser Phe Trp Val Ser Met Ile Ser Tyr Asn Ser LeuGly 295 300 305 aag tct cca gtg gcc acc ctg agg att cca gct att caa gaaaaa tca 1136 Lys Ser Pro Val Ala Thr Leu Arg Ile Pro Ala Ile Gln Glu LysSer 310 315 320 325 ttt cag tgc att gag gtc atg cag gcc tgc gtt gct gaggac cag cta 1184 Phe Gln Cys Ile Glu Val Met Gln Ala Cys Val Ala Glu AspGln Leu 330 335 340 gtg gtg aag tgg caa agc tct gct cta gac gtg aac acttgg atg att 1232 Val Val Lys Trp Gln Ser Ser Ala Leu Asp Val Asn Thr TrpMet Ile 345 350 355 gaa tgg ttt ccg gat gtg gac tca gag ccc acc acc ctttcc tgg gaa 1280 Glu Trp Phe Pro Asp Val Asp Ser Glu Pro Thr Thr Leu SerTrp Glu 360 365 370 tct gtg tct cag gcc acg aac tgg acg atc cag caa gataaa tta aaa 1328 Ser Val Ser Gln Ala Thr Asn Trp Thr Ile Gln Gln Asp LysLeu Lys 375 380 385 cct ttc tgg tgc tat aac atc tct gtg tat cca atg ttgcat gac aaa 1376 Pro Phe Trp Cys Tyr Asn Ile Ser Val Tyr Pro Met Leu HisAsp Lys 390 395 400 405 gtt ggc gag cca tat tcc atc cag gct tat gcc aaagaa ggc gtt cca 1424 Val Gly Glu Pro Tyr Ser Ile Gln Ala Tyr Ala Lys GluGly Val Pro 410 415 420 tca gaa ggt cct gag acc aag gtg gag aac att ggcgtg aag acg gtc 1472 Ser Glu Gly Pro Glu Thr Lys Val Glu Asn Ile Gly ValLys Thr Val 425 430 435 acg atc aca tgg aaa gag att ccc aag agt gag agaaag ggt atc atc 1520 Thr Ile Thr Trp Lys Glu Ile Pro Lys Ser Glu Arg LysGly Ile Ile 440 445 450 tgc aac tac acc atc ttt tac caa gct gaa ggt ggaaaa gga ttc tcc 1568 Cys Asn Tyr Thr Ile Phe Tyr Gln Ala Glu Gly Gly LysGly Phe Ser 455 460 465 aag aca gtc aat tcc agc atc ttg cag tac ggc ctggag tcc ctg aaa 1616 Lys Thr Val Asn Ser Ser Ile Leu Gln Tyr Gly Leu GluSer Leu Lys 470 475 480 485 cga aag acc tct tac att gtt cag gtc atg gccagc acc agt gct ggg 1664 Arg Lys Thr Ser Tyr Ile Val Gln Val Met Ala SerThr Ser Ala Gly 490 495 500 gga acc aac ggg acc agc ata aat ttc aag acattg tca ttc agt gtc 1712 Gly Thr Asn Gly Thr Ser Ile Asn Phe Lys Thr LeuSer Phe Ser Val 505 510 515 ttt gag att atc ctc ata act tct ctg att ggtgga ggc ctt ctt att 1760 Phe Glu Ile Ile Leu Ile Thr Ser Leu Ile Gly GlyGly Leu Leu Ile 520 525 530 ctc att atc ctg aca gtg gca tat ggt ctc aaaaaa ccc aac aaa ttg 1808 Leu Ile Ile Leu Thr Val Ala Tyr Gly Leu Lys LysPro Asn Lys Leu 535 540 545 act cat ctg tgt tgg ccc acc gtt ccc aac cctgct gaa agt agt ata 1856 Thr His Leu Cys Trp Pro Thr Val Pro Asn Pro AlaGlu Ser Ser Ile 550 555 560 565 gcc aca tgg cat gga gat gat ttc aag gataag cta aac ctg aag gag 1904 Ala Thr Trp His Gly Asp Asp Phe Lys Asp LysLeu Asn Leu Lys Glu 570 575 580 tct gat gac tct gtg aac aca gaa gac aggatc tta aaa cca tgt tcc 1952 Ser Asp Asp Ser Val Asn Thr Glu Asp Arg IleLeu Lys Pro Cys Ser 585 590 595 acc ccc agt gac aag ttg gtg att gac aagttg gtg gtg aac ttt ggg 2000 Thr Pro Ser Asp Lys Leu Val Ile Asp Lys LeuVal Val Asn Phe Gly 600 605 610 aat gtt ctg caa gaa att ttc aca gat gaagcc aga acg ggt cag gaa 2048 Asn Val Leu Gln Glu Ile Phe Thr Asp Glu AlaArg Thr Gly Gln Glu 615 620 625 aac aat tta gga ggg gaa aag aat ggg actaga att ctg tct tcc tgc 2096 Asn Asn Leu Gly Gly Glu Lys Asn Gly Thr ArgIle Leu Ser Ser Cys 630 635 640 645 cca act tca ata taagtgtggactaaaatgcg agaaaggtgt cctgtggtct 2148 Pro Thr Ser Ile atgcaaattagaaaggacat gcagagtttt ccaactagga agactgaatc tgtggcccca 2208 agagaaccatctctgaagac tgggtatgtg gtcttttcca cacatggacc acctacggat 2268 gcaatctgtaatgcatgtgc atgagaagtc tgttattaag tagagtgtga aaacatggtt 2328 atggtaataggaacagcttt taaaatgctt ttgtatttgg gcctttcata caaaaaagcc 2388 ataataccattttcatgtaa tgctatactt ctatactatt ttcatgtaat actatacttc 2448 tatactattttcatgtaata ctatacttct atactatttt catgtaatac tatacttcta 2508 tattaaagttttacccactc a 2529 46 649 PRT Homo sapiens 46 Met Met Trp Thr Trp Ala LeuTrp Met Leu Pro Ser Leu Cys Lys Phe 1 5 10 15 Ser Leu Ala Ala Leu ProAla Lys Pro Glu Asn Ile Ser Cys Val Tyr 20 25 30 Tyr Tyr Arg Lys Asn LeuThr Cys Thr Trp Ser Pro Gly Lys Glu Thr 35 40 45 Ser Tyr Thr Gln Tyr ThrVal Lys Arg Thr Tyr Ala Phe Gly Glu Lys 50 55 60 His Asp Asn Cys Thr ThrAsn Ser Ser Thr Ser Glu Asn Arg Ala Ser 65 70 75 80 Cys Ser Phe Phe LeuPro Arg Ile Thr Ile Pro Asp Asn Tyr Thr Ile 85 90 95 Glu Val Glu Ala GluAsn Gly Asp Gly Val Ile Lys Ser His Met Thr 100 105 110 Tyr Trp Arg LeuGlu Asn Ile Ala Lys Thr Glu Pro Pro Lys Ile Phe 115 120 125 Arg Val LysPro Val Leu Gly Ile Lys Arg Met Ile Gln Ile Glu Trp 130 135 140 Ile LysPro Glu Leu Ala Pro Val Ser Ser Asp Leu Lys Tyr Thr Leu 145 150 155 160Arg Phe Arg Thr Val Asn Ser Thr Ser Trp Met Glu Val Asn Phe Ala 165 170175 Lys Asn Arg Lys Asp Lys Asn Gln Thr Tyr Asn Leu Thr Gly Leu Gln 180185 190 Pro Phe Thr Glu Tyr Val Ile Ala Leu Arg Cys Ala Val Lys Glu Ser195 200 205 Lys Phe Trp Ser Asp Trp Ser Gln Glu Lys Met Gly Met Thr GluGlu 210 215 220 Glu Ala Pro Cys Gly Leu Glu Leu Trp Arg Val Leu Lys ProAla Glu 225 230 235 240 Ala Asp Gly Arg Arg Pro Val Arg Leu Leu Trp LysLys Ala Arg Gly 245 250 255 Ala Pro Val Leu Glu Lys Thr Leu Gly Tyr AsnIle Trp Tyr Tyr Pro 260 265 270 Glu Ser Asn Thr Asn Leu Thr Glu Thr MetAsn Thr Thr Asn Gln Gln 275 280 285 Leu Glu Leu His Leu Gly Gly Glu SerPhe Trp Val Ser Met Ile Ser 290 295 300 Tyr Asn Ser Leu Gly Lys Ser ProVal Ala Thr Leu Arg Ile Pro Ala 305 310 315 320 Ile Gln Glu Lys Ser PheGln Cys Ile Glu Val Met Gln Ala Cys Val 325 330 335 Ala Glu Asp Gln LeuVal Val Lys Trp Gln Ser Ser Ala Leu Asp Val 340 345 350 Asn Thr Trp MetIle Glu Trp Phe Pro Asp Val Asp Ser Glu Pro Thr 355 360 365 Thr Leu SerTrp Glu Ser Val Ser Gln Ala Thr Asn Trp Thr Ile Gln 370 375 380 Gln AspLys Leu Lys Pro Phe Trp Cys Tyr Asn Ile Ser Val Tyr Pro 385 390 395 400Met Leu His Asp Lys Val Gly Glu Pro Tyr Ser Ile Gln Ala Tyr Ala 405 410415 Lys Glu Gly Val Pro Ser Glu Gly Pro Glu Thr Lys Val Glu Asn Ile 420425 430 Gly Val Lys Thr Val Thr Ile Thr Trp Lys Glu Ile Pro Lys Ser Glu435 440 445 Arg Lys Gly Ile Ile Cys Asn Tyr Thr Ile Phe Tyr Gln Ala GluGly 450 455 460 Gly Lys Gly Phe Ser Lys Thr Val Asn Ser Ser Ile Leu GlnTyr Gly 465 470 475 480 Leu Glu Ser Leu Lys Arg Lys Thr Ser Tyr Ile ValGln Val Met Ala 485 490 495 Ser Thr Ser Ala Gly Gly Thr Asn Gly Thr SerIle Asn Phe Lys Thr 500 505 510 Leu Ser Phe Ser Val Phe Glu Ile Ile LeuIle Thr Ser Leu Ile Gly 515 520 525 Gly Gly Leu Leu Ile Leu Ile Ile LeuThr Val Ala Tyr Gly Leu Lys 530 535 540 Lys Pro Asn Lys Leu Thr His LeuCys Trp Pro Thr Val Pro Asn Pro 545 550 555 560 Ala Glu Ser Ser Ile AlaThr Trp His Gly Asp Asp Phe Lys Asp Lys 565 570 575 Leu Asn Leu Lys GluSer Asp Asp Ser Val Asn Thr Glu Asp Arg Ile 580 585 590 Leu Lys Pro CysSer Thr Pro Ser Asp Lys Leu Val Ile Asp Lys Leu 595 600 605 Val Val AsnPhe Gly Asn Val Leu Gln Glu Ile Phe Thr Asp Glu Ala 610 615 620 Arg ThrGly Gln Glu Asn Asn Leu Gly Gly Glu Lys Asn Gly Thr Arg 625 630 635 640Ile Leu Ser Ser Cys Pro Thr Ser Ile 645 47 1947 DNA Artificial SequenceDegenerate polynucleotide sequence of SEQ ID NO46 47 atgatgtggacntgggcnyt ntggatgytn ccnwsnytnt gyaarttyws nytngcngcn 60 ytnccngcnaarccngaraa yathwsntgy gtntaytayt aymgnaaraa yytnacntgy 120 acntggwsnccnggnaarga racnwsntay acncartaya cngtnaarmg nacntaygcn 180 ttyggngaraarcaygayaa ytgyacnacn aaywsnwsna cnwsngaraa ymgngcnwsn 240 tgywsnttyttyytnccnmg nathacnath ccngayaayt ayacnathga rgtngargcn 300 garaayggngayggngtnat haarwsncay atgacntayt ggmgnytnga raayathgcn 360 aaracngarccnccnaarat httymgngtn aarccngtny tnggnathaa rmgnatgath 420 carathgartggathaarcc ngarytngcn ccngtnwsnw sngayytnaa rtayacnytn 480 mgnttymgnacngtnaayws nacnwsntgg atggargtna ayttygcnaa raaymgnaar 540 gayaaraaycaracntayaa yytnacnggn ytncarccnt tyacngarta ygtnathgcn 600 ytnmgntgygcngtnaarga rwsnaartty tggwsngayt ggwsncarga raaratgggn 660 atgacngargargargcncc ntgyggnytn garytntggm gngtnytnaa rccngcngar 720 gcngayggnmgnmgnccngt nmgnytnytn tggaaraarg cnmgnggngc nccngtnytn 780 garaaracnytnggntayaa yathtggtay tayccngarw snaayacnaa yytnacngar 840 acnatgaayacnacnaayca rcarytngar ytncayytng gnggngarws nttytgggtn 900 wsnatgathwsntayaayws nytnggnaar wsnccngtng cnacnytnmg nathccngcn 960 athcargaraarwsnttyca rtgyathgar gtnatgcarg cntgygtngc ngargaycar 1020 ytngtngtnaartggcarws nwsngcnytn gaygtnaaya cntggatgat hgartggtty 1080 ccngaygtngaywsngarcc nacnacnytn wsntgggarw sngtnwsnca rgcnacnaay 1140 tggacnathcarcargayaa rytnaarccn ttytggtgyt ayaayathws ngtntayccn 1200 atgytncaygayaargtngg ngarccntay wsnathcarg cntaygcnaa rgarggngtn 1260 ccnwsngarggnccngarac naargtngar aayathggng tnaaracngt nacnathacn 1320 tggaargarathccnaarws ngarmgnaar ggnathatht gyaaytayac nathttytay 1380 cargcngarggnggnaargg nttywsnaar acngtnaayw snwsnathyt ncartayggn 1440 ytngarwsnytnaarmgnaa racnwsntay athgtncarg tnatggcnws nacnwsngcn 1500 ggnggnacnaayggnacnws nathaaytty aaracnytnw snttywsngt nttygarath 1560 athytnathacnwsnytnat hggnggnggn ytnytnathy tnathathyt nacngtngcn 1620 tayggnytnaaraarccnaa yaarytnacn cayytntgyt ggccnacngt nccnaayccn 1680 gcngarwsnwsnathgcnac ntggcayggn gaygayttya argayaaryt naayytnaar 1740 garwsngaygaywsngtnaa yacngargay mgnathytna arccntgyws nacnccnwsn 1800 gayaarytngtnathgayaa rytngtngtn aayttyggna aygtnytnca rgarathtty 1860 acngaygargcnmgnacngg ncargaraay aayytnggng gngaraaraa yggnacnmgn 1920 athytnwsnwsntgyccnac nwsnath 1947 48 32 PRT Homo sapiens 48 Met Cys Ile Arg GlnLeu Lys Phe Phe Thr Thr Ala Cys Val Cys Glu 1 5 10 15 Cys Pro Gln AsnIle Leu Ser Pro Gln Pro Ser Cys Val Asn Leu Gly 20 25 30 49 23 DNAArtificial Sequence Oligonucloetide primer ZC21195 49 gaggagaccataacccccga cag 23 50 23 DNA Artificial Sequence Oligonucloetide primerZC21196 50 catagctccc accacacgat ttt 23 51 23 DNA Artificial SequenceOligonucleotide primer ZC27900 51 gccccgtgag gttgtacgtt tgg 23 52 13 PRTHomo sapiens 52 Met Lys Leu Ser Pro Gln Pro Ser Cys Val Asn Leu Gly 1 510 53 2903 DNA Homo sapiens CDS (497)...(2482) 53 tgaaaagaca tgtgtgtgcagtatgaaaat tgagacagga aggcagagtg tcagcttgtt 60 ccacctcagc tgggaatgtgcatcaggcaa ctcaagtttt tcaccacggc atgtgtctgt 120 gaatgtccgc aaaacattagtttcactctt gtcgccaggt tggagtacaa tggcacgatc 180 ttggctcact gcaacctctgcctcccgggt tcaagcgatt ctcctgcctc agcctcccga 240 gtagctggga ttacagttaacaataatgca atccatttcc cagcataagt gggtaagtgc 300 cactttgact tgggctgggcttaaaagcac aagaaaagct cgcagacaat cagagtggaa 360 acactcccac atcttagtgtggataaatta aagtccagat tgttcttcct gtcctgactt 420 gtgctgtggg aggtggagttgcctttgatg caaatccttt gagccagcag aacatctgtg 480 gaacatcccc tgatac atgaag ctc tct ccc cag cct tca tgt gtt aac ctg 532 Met Lys Leu Ser Pro GlnPro Ser Cys Val Asn Leu 1 5 10 ggg atg atg tgg acc tgg gca ctg tgg atgctc cct tca ctc tgc aaa 580 Gly Met Met Trp Thr Trp Ala Leu Trp Met LeuPro Ser Leu Cys Lys 15 20 25 ttc agc ctg gca gct ctg cca gct aag cct gagaac att tcc tgt gtc 628 Phe Ser Leu Ala Ala Leu Pro Ala Lys Pro Glu AsnIle Ser Cys Val 30 35 40 tac tac tat agg aaa aat tta acc tgc act tgg agtcca gga aag gaa 676 Tyr Tyr Tyr Arg Lys Asn Leu Thr Cys Thr Trp Ser ProGly Lys Glu 45 50 55 60 acc agt tat acc cag tac aca gtt aag aga act tacgct ttt gga gaa 724 Thr Ser Tyr Thr Gln Tyr Thr Val Lys Arg Thr Tyr AlaPhe Gly Glu 65 70 75 aaa cat gat aat tgt aca acc aat agt tct aca agt gaaaat cgt gct 772 Lys His Asp Asn Cys Thr Thr Asn Ser Ser Thr Ser Glu AsnArg Ala 80 85 90 tcg tgc tct ttt ttc ctt cca aga ata acg atc cca gat aattat acc 820 Ser Cys Ser Phe Phe Leu Pro Arg Ile Thr Ile Pro Asp Asn TyrThr 95 100 105 att gag gtg gaa gct gaa aat gga gat ggt gta att aaa tctcat atg 868 Ile Glu Val Glu Ala Glu Asn Gly Asp Gly Val Ile Lys Ser HisMet 110 115 120 aca tac tgg aga tta gag aac ata gcg aaa act gaa cca cctaag att 916 Thr Tyr Trp Arg Leu Glu Asn Ile Ala Lys Thr Glu Pro Pro LysIle 125 130 135 140 ttc cgt gtg aaa cca gtt ttg ggc atc aaa cga atg attcaa att gaa 964 Phe Arg Val Lys Pro Val Leu Gly Ile Lys Arg Met Ile GlnIle Glu 145 150 155 tgg ata aag cct gag ttg gcg cct gtt tca tct gat ttaaaa tac aca 1012 Trp Ile Lys Pro Glu Leu Ala Pro Val Ser Ser Asp Leu LysTyr Thr 160 165 170 ctt cga ttc agg aca gtc aac agt acc agc tgg atg gaagtc aac ttc 1060 Leu Arg Phe Arg Thr Val Asn Ser Thr Ser Trp Met Glu ValAsn Phe 175 180 185 gct aag aac cgt aag gat aaa aac caa acg tac aac ctcacg ggg ctg 1108 Ala Lys Asn Arg Lys Asp Lys Asn Gln Thr Tyr Asn Leu ThrGly Leu 190 195 200 cag cct ttt aca gaa tat gtc ata gct ctg cga tgt gcggtc aag gag 1156 Gln Pro Phe Thr Glu Tyr Val Ile Ala Leu Arg Cys Ala ValLys Glu 205 210 215 220 tca aag ttc tgg agt gac tgg agc caa gaa aaa atggga atg act gag 1204 Ser Lys Phe Trp Ser Asp Trp Ser Gln Glu Lys Met GlyMet Thr Glu 225 230 235 gaa gaa gct cca tgt ggc ctg gaa ctg tgg aga gtcctg aaa cca gct 1252 Glu Glu Ala Pro Cys Gly Leu Glu Leu Trp Arg Val LeuLys Pro Ala 240 245 250 gag gcg gat gga aga agg cca gtg cgg ttg tta tggaag aag gca aga 1300 Glu Ala Asp Gly Arg Arg Pro Val Arg Leu Leu Trp LysLys Ala Arg 255 260 265 gga gcc cca gtc cta gag aaa aca ctt ggc tac aacata tgg tac tat 1348 Gly Ala Pro Val Leu Glu Lys Thr Leu Gly Tyr Asn IleTrp Tyr Tyr 270 275 280 cca gaa agc aac act aac ctc aca gaa aca atg aacact act aac cag 1396 Pro Glu Ser Asn Thr Asn Leu Thr Glu Thr Met Asn ThrThr Asn Gln 285 290 295 300 cag ctt gaa ctg cat ctg gga ggc gag agc ttttgg gtg tct atg att 1444 Gln Leu Glu Leu His Leu Gly Gly Glu Ser Phe TrpVal Ser Met Ile 305 310 315 tct tat aat tct ctt ggg aag tct cca gtg gccacc ctg agg att cca 1492 Ser Tyr Asn Ser Leu Gly Lys Ser Pro Val Ala ThrLeu Arg Ile Pro 320 325 330 gct att caa gaa aaa tca ttt cag tgc att gaggtc atg cag gcc tgc 1540 Ala Ile Gln Glu Lys Ser Phe Gln Cys Ile Glu ValMet Gln Ala Cys 335 340 345 gtt gct gag gac cag cta gtg gtg aag tgg caaagc tct gct cta gac 1588 Val Ala Glu Asp Gln Leu Val Val Lys Trp Gln SerSer Ala Leu Asp 350 355 360 gtg aac act tgg atg att gaa tgg ttt ccg gatgtg gac tca gag ccc 1636 Val Asn Thr Trp Met Ile Glu Trp Phe Pro Asp ValAsp Ser Glu Pro 365 370 375 380 acc acc ctt tcc tgg gaa tct gtg tct caggcc acg aac tgg acg atc 1684 Thr Thr Leu Ser Trp Glu Ser Val Ser Gln AlaThr Asn Trp Thr Ile 385 390 395 cag caa gat aaa tta aaa cct ttc tgg tgctat aac atc tct gtg tat 1732 Gln Gln Asp Lys Leu Lys Pro Phe Trp Cys TyrAsn Ile Ser Val Tyr 400 405 410 cca atg ttg cat gac aaa gtt ggc gag ccatat tcc atc cag gct tat 1780 Pro Met Leu His Asp Lys Val Gly Glu Pro TyrSer Ile Gln Ala Tyr 415 420 425 gcc aaa gaa ggc gtt cca tca gaa ggt cctgag acc aag gtg gag aac 1828 Ala Lys Glu Gly Val Pro Ser Glu Gly Pro GluThr Lys Val Glu Asn 430 435 440 att ggc gtg aag acg gtc acg atc aca tggaaa gag att ccc aag agt 1876 Ile Gly Val Lys Thr Val Thr Ile Thr Trp LysGlu Ile Pro Lys Ser 445 450 455 460 gag aga aag ggt atc atc tgc aac tacacc atc ttt tac caa gct gaa 1924 Glu Arg Lys Gly Ile Ile Cys Asn Tyr ThrIle Phe Tyr Gln Ala Glu 465 470 475 ggt gga aaa gga ttc tcc aag aca gtcaat tcc agc atc ttg cag tac 1972 Gly Gly Lys Gly Phe Ser Lys Thr Val AsnSer Ser Ile Leu Gln Tyr 480 485 490 ggc ctg gag tcc ctg aaa cga aag acctct tac att gtt cag gtc atg 2020 Gly Leu Glu Ser Leu Lys Arg Lys Thr SerTyr Ile Val Gln Val Met 495 500 505 gcc agc acc agt gct ggg gga acc aacggg acc agc ata aat ttc aag 2068 Ala Ser Thr Ser Ala Gly Gly Thr Asn GlyThr Ser Ile Asn Phe Lys 510 515 520 aca ttg tca ttc agt gtc ttt gag attatc ctc ata act tct ctg att 2116 Thr Leu Ser Phe Ser Val Phe Glu Ile IleLeu Ile Thr Ser Leu Ile 525 530 535 540 ggt gga ggc ctt ctt att ctc attatc ctg aca gtg gca tat ggt ctc 2164 Gly Gly Gly Leu Leu Ile Leu Ile IleLeu Thr Val Ala Tyr Gly Leu 545 550 555 aaa aaa ccc aac aaa ttg act catctg tgt tgg ccc acc gtt ccc aac 2212 Lys Lys Pro Asn Lys Leu Thr His LeuCys Trp Pro Thr Val Pro Asn 560 565 570 cct gct gaa agt agt ata gcc acatgg cat gga gat gat ttc aag gat 2260 Pro Ala Glu Ser Ser Ile Ala Thr TrpHis Gly Asp Asp Phe Lys Asp 575 580 585 aag cta aac ctg aag gag tct gatgac tct gtg aac aca gaa gac agg 2308 Lys Leu Asn Leu Lys Glu Ser Asp AspSer Val Asn Thr Glu Asp Arg 590 595 600 atc tta aaa cca tgt tcc acc cccagt gac aag ttg gtg att gac aag 2356 Ile Leu Lys Pro Cys Ser Thr Pro SerAsp Lys Leu Val Ile Asp Lys 605 610 615 620 ttg gtg gtg aac ttt ggg aatgtt ctg caa gaa att ttc aca gat gaa 2404 Leu Val Val Asn Phe Gly Asn ValLeu Gln Glu Ile Phe Thr Asp Glu 625 630 635 gcc aga acg ggt cag gaa aacaat tta gga ggg gaa aag aat ggg act 2452 Ala Arg Thr Gly Gln Glu Asn AsnLeu Gly Gly Glu Lys Asn Gly Thr 640 645 650 aga att ctg tct tcc tgc ccaact tca ata taagtgtgga ctaaaatgcg 2502 Arg Ile Leu Ser Ser Cys Pro ThrSer Ile 655 660 agaaaggtgt cctgtggtct atgcaaatta gaaaggacat gcagagttttccaactagga 2562 agactgaatc tgtggcccca agagaaccat ctctgaagac tgggtatgtggtcttttcca 2622 cacatggacc acctacggat gcaatctgta atgcatgtgc atgagaagtctgttattaag 2682 tagagtgtga aaacatggtt atggtaatag gaacagcttt taaaatgcttttgtatttgg 2742 gcctttcata caaaaaagcc ataataccat tttcatgtaa tgctatacttctatactatt 2802 ttcatgtaat actatacttc tatactattt tcatgtaata ctatacttctatactatttt 2862 catgtaatac tatacttcta tattaaagtt ttacccactc a 2903 54662 PRT Homo sapiens 54 Met Lys Leu Ser Pro Gln Pro Ser Cys Val Asn LeuGly Met Met Trp 1 5 10 15 Thr Trp Ala Leu Trp Met Leu Pro Ser Leu CysLys Phe Ser Leu Ala 20 25 30 Ala Leu Pro Ala Lys Pro Glu Asn Ile Ser CysVal Tyr Tyr Tyr Arg 35 40 45 Lys Asn Leu Thr Cys Thr Trp Ser Pro Gly LysGlu Thr Ser Tyr Thr 50 55 60 Gln Tyr Thr Val Lys Arg Thr Tyr Ala Phe GlyGlu Lys His Asp Asn 65 70 75 80 Cys Thr Thr Asn Ser Ser Thr Ser Glu AsnArg Ala Ser Cys Ser Phe 85 90 95 Phe Leu Pro Arg Ile Thr Ile Pro Asp AsnTyr Thr Ile Glu Val Glu 100 105 110 Ala Glu Asn Gly Asp Gly Val Ile LysSer His Met Thr Tyr Trp Arg 115 120 125 Leu Glu Asn Ile Ala Lys Thr GluPro Pro Lys Ile Phe Arg Val Lys 130 135 140 Pro Val Leu Gly Ile Lys ArgMet Ile Gln Ile Glu Trp Ile Lys Pro 145 150 155 160 Glu Leu Ala Pro ValSer Ser Asp Leu Lys Tyr Thr Leu Arg Phe Arg 165 170 175 Thr Val Asn SerThr Ser Trp Met Glu Val Asn Phe Ala Lys Asn Arg 180 185 190 Lys Asp LysAsn Gln Thr Tyr Asn Leu Thr Gly Leu Gln Pro Phe Thr 195 200 205 Glu TyrVal Ile Ala Leu Arg Cys Ala Val Lys Glu Ser Lys Phe Trp 210 215 220 SerAsp Trp Ser Gln Glu Lys Met Gly Met Thr Glu Glu Glu Ala Pro 225 230 235240 Cys Gly Leu Glu Leu Trp Arg Val Leu Lys Pro Ala Glu Ala Asp Gly 245250 255 Arg Arg Pro Val Arg Leu Leu Trp Lys Lys Ala Arg Gly Ala Pro Val260 265 270 Leu Glu Lys Thr Leu Gly Tyr Asn Ile Trp Tyr Tyr Pro Glu SerAsn 275 280 285 Thr Asn Leu Thr Glu Thr Met Asn Thr Thr Asn Gln Gln LeuGlu Leu 290 295 300 His Leu Gly Gly Glu Ser Phe Trp Val Ser Met Ile SerTyr Asn Ser 305 310 315 320 Leu Gly Lys Ser Pro Val Ala Thr Leu Arg IlePro Ala Ile Gln Glu 325 330 335 Lys Ser Phe Gln Cys Ile Glu Val Met GlnAla Cys Val Ala Glu Asp 340 345 350 Gln Leu Val Val Lys Trp Gln Ser SerAla Leu Asp Val Asn Thr Trp 355 360 365 Met Ile Glu Trp Phe Pro Asp ValAsp Ser Glu Pro Thr Thr Leu Ser 370 375 380 Trp Glu Ser Val Ser Gln AlaThr Asn Trp Thr Ile Gln Gln Asp Lys 385 390 395 400 Leu Lys Pro Phe TrpCys Tyr Asn Ile Ser Val Tyr Pro Met Leu His 405 410 415 Asp Lys Val GlyGlu Pro Tyr Ser Ile Gln Ala Tyr Ala Lys Glu Gly 420 425 430 Val Pro SerGlu Gly Pro Glu Thr Lys Val Glu Asn Ile Gly Val Lys 435 440 445 Thr ValThr Ile Thr Trp Lys Glu Ile Pro Lys Ser Glu Arg Lys Gly 450 455 460 IleIle Cys Asn Tyr Thr Ile Phe Tyr Gln Ala Glu Gly Gly Lys Gly 465 470 475480 Phe Ser Lys Thr Val Asn Ser Ser Ile Leu Gln Tyr Gly Leu Glu Ser 485490 495 Leu Lys Arg Lys Thr Ser Tyr Ile Val Gln Val Met Ala Ser Thr Ser500 505 510 Ala Gly Gly Thr Asn Gly Thr Ser Ile Asn Phe Lys Thr Leu SerPhe 515 520 525 Ser Val Phe Glu Ile Ile Leu Ile Thr Ser Leu Ile Gly GlyGly Leu 530 535 540 Leu Ile Leu Ile Ile Leu Thr Val Ala Tyr Gly Leu LysLys Pro Asn 545 550 555 560 Lys Leu Thr His Leu Cys Trp Pro Thr Val ProAsn Pro Ala Glu Ser 565 570 575 Ser Ile Ala Thr Trp His Gly Asp Asp PheLys Asp Lys Leu Asn Leu 580 585 590 Lys Glu Ser Asp Asp Ser Val Asn ThrGlu Asp Arg Ile Leu Lys Pro 595 600 605 Cys Ser Thr Pro Ser Asp Lys LeuVal Ile Asp Lys Leu Val Val Asn 610 615 620 Phe Gly Asn Val Leu Gln GluIle Phe Thr Asp Glu Ala Arg Thr Gly 625 630 635 640 Gln Glu Asn Asn LeuGly Gly Glu Lys Asn Gly Thr Arg Ile Leu Ser 645 650 655 Ser Cys Pro ThrSer Ile 660 55 1986 DNA Artificial Sequence Degenerate polynucleotidesequence of SEQ ID NO54 55 atgaarytnw snccncarcc nwsntgygtn aayytnggnatgatgtggac ntgggcnytn 60 tggatgytnc cnwsnytntg yaarttywsn ytngcngcnytnccngcnaa rccngaraay 120 athwsntgyg tntaytayta ymgnaaraay ytnacntgyacntggwsncc nggnaargar 180 acnwsntaya cncartayac ngtnaarmgn acntaygcnttyggngaraa rcaygayaay 240 tgyacnacna aywsnwsnac nwsngaraay mgngcnwsntgywsnttytt yytnccnmgn 300 athacnathc cngayaayta yacnathgar gtngargcngaraayggnga yggngtnath 360 aarwsncaya tgacntaytg gmgnytngar aayathgcnaaracngarcc nccnaarath 420 ttymgngtna arccngtnyt nggnathaar mgnatgathcarathgartg gathaarccn 480 garytngcnc cngtnwsnws ngayytnaar tayacnytnmgnttymgnac ngtnaaywsn 540 acnwsntgga tggargtnaa yttygcnaar aaymgnaargayaaraayca racntayaay 600 ytnacnggny tncarccntt yacngartay gtnathgcnytnmgntgygc ngtnaargar 660 wsnaarttyt ggwsngaytg gwsncargar aaratgggnatgacngarga rgargcnccn 720 tgyggnytng arytntggmg ngtnytnaar ccngcngargcngayggnmg nmgnccngtn 780 mgnytnytnt ggaaraargc nmgnggngcn ccngtnytngaraaracnyt nggntayaay 840 athtggtayt ayccngarws naayacnaay ytnacngaracnatgaayac nacnaaycar 900 carytngary tncayytngg nggngarwsn ttytgggtnwsnatgathws ntayaaywsn 960 ytnggnaarw snccngtngc nacnytnmgn athccngcnathcargaraa rwsnttycar 1020 tgyathgarg tnatgcargc ntgygtngcn gargaycarytngtngtnaa rtggcarwsn 1080 wsngcnytng aygtnaayac ntggatgath gartggttyccngaygtnga ywsngarccn 1140 acnacnytnw sntgggarws ngtnwsncar gcnacnaaytggacnathca rcargayaar 1200 ytnaarccnt tytggtgyta yaayathwsn gtntayccnatgytncayga yaargtnggn 1260 garccntayw snathcargc ntaygcnaar garggngtnccnwsngargg nccngaracn 1320 aargtngara ayathggngt naaracngtn acnathacntggaargarat hccnaarwsn 1380 garmgnaarg gnathathtg yaaytayacn athttytaycargcngargg nggnaarggn 1440 ttywsnaara cngtnaayws nwsnathytn cartayggnytngarwsnyt naarmgnaar 1500 acnwsntaya thgtncargt natggcnwsn acnwsngcnggnggnacnaa yggnacnwsn 1560 athaayttya aracnytnws nttywsngtn ttygarathathytnathac nwsnytnath 1620 ggnggnggny tnytnathyt nathathytn acngtngcntayggnytnaa raarccnaay 1680 aarytnacnc ayytntgytg gccnacngtn ccnaayccngcngarwsnws nathgcnacn 1740 tggcayggng aygayttyaa rgayaarytn aayytnaargarwsngayga ywsngtnaay 1800 acngargaym gnathytnaa rccntgywsn acnccnwsngayaarytngt nathgayaar 1860 ytngtngtna ayttyggnaa ygtnytncar garathttyacngaygargc nmgnacnggn 1920 cargaraaya ayytnggngg ngaraaraay ggnacnmgnathytnwsnws ntgyccnacn 1980 wsnath 1986 56 2748 DNA mus musculus CDS(237)...(2222) 56 gatggggccc tgaatgttga tctgacagaa ttccagacca acctggtggttattgtcctt 60 ttcatctggt catgctgaat atactctcaa gatgtgctgg agaaggtgctgctgtccggg 120 ctctcagaga aggcagtgct ggaggcgttc ctggcccggg tctcctcctactgttcctgg 180 tagcccagcc ttctcggggt ggaaggagaa gctggccagg tgagctctgaggaagc atg 239 Met 1 ctg agc agc cag aag gga tcc tgc agc cag gaa cca ggggca gcc cac 287 Leu Ser Ser Gln Lys Gly Ser Cys Ser Gln Glu Pro Gly AlaAla His 5 10 15 gtc cag cct ctg ggt gtg aac gct gga ata atg tgg acc ttggca ctg 335 Val Gln Pro Leu Gly Val Asn Ala Gly Ile Met Trp Thr Leu AlaLeu 20 25 30 tgg gca ttc tct ttc ctc tgc aaa ttc agc ctg gca gtc ctg ccgact 383 Trp Ala Phe Ser Phe Leu Cys Lys Phe Ser Leu Ala Val Leu Pro Thr35 40 45 aag cca gag aac att tcc tgc gtc ttt tac ttc gac aga aat ctg act431 Lys Pro Glu Asn Ile Ser Cys Val Phe Tyr Phe Asp Arg Asn Leu Thr 5055 60 65 tgc act tgg aga cca gag aag gaa acc aat gat acc agc tac att gtg479 Cys Thr Trp Arg Pro Glu Lys Glu Thr Asn Asp Thr Ser Tyr Ile Val 7075 80 act ttg act tac tcc tat gga aaa agc aat tat agt gac aat gct aca527 Thr Leu Thr Tyr Ser Tyr Gly Lys Ser Asn Tyr Ser Asp Asn Ala Thr 8590 95 gag gct tca tat tct ttt ccc cgt tcc tgt gca atg ccc cca gac atc575 Glu Ala Ser Tyr Ser Phe Pro Arg Ser Cys Ala Met Pro Pro Asp Ile 100105 110 tgc agt gtt gaa gta caa gct caa aat gga gat ggt aaa gtt aaa tct623 Cys Ser Val Glu Val Gln Ala Gln Asn Gly Asp Gly Lys Val Lys Ser 115120 125 gac atc aca tat tgg cat tta atc tcc ata gca aaa acc gaa cca cct671 Asp Ile Thr Tyr Trp His Leu Ile Ser Ile Ala Lys Thr Glu Pro Pro 130135 140 145 ata att tta agt gtg aat cca att tgt aat aga atg ttc cag atacaa 719 Ile Ile Leu Ser Val Asn Pro Ile Cys Asn Arg Met Phe Gln Ile Gln150 155 160 tgg aaa ccg cgt gaa aag act cgt ggg ttt cct tta gta tgc atgctt 767 Trp Lys Pro Arg Glu Lys Thr Arg Gly Phe Pro Leu Val Cys Met Leu165 170 175 cgg ttc aga act gtc aac agt agc cgc tgg acg gaa gtc aat tttgaa 815 Arg Phe Arg Thr Val Asn Ser Ser Arg Trp Thr Glu Val Asn Phe Glu180 185 190 aac tgt aaa cag gtc tgc aac ctc aca gga ctt cag gct ttc acagaa 863 Asn Cys Lys Gln Val Cys Asn Leu Thr Gly Leu Gln Ala Phe Thr Glu195 200 205 tat gtc ctg gct cta cga ttc agg ttc aat gac tca aga tat tggagc 911 Tyr Val Leu Ala Leu Arg Phe Arg Phe Asn Asp Ser Arg Tyr Trp Ser210 215 220 225 aag tgg agc aaa gaa gaa acc aga gtg act atg gag gaa gttcca cat 959 Lys Trp Ser Lys Glu Glu Thr Arg Val Thr Met Glu Glu Val ProHis 230 235 240 gtc ctg gac ctg tgg aga att ctg gaa cca gca gac atg aacgga gac 1007 Val Leu Asp Leu Trp Arg Ile Leu Glu Pro Ala Asp Met Asn GlyAsp 245 250 255 agg aag gtg cga ttg ctg tgg aag aag gca aga gga gcc cccgtc ttg 1055 Arg Lys Val Arg Leu Leu Trp Lys Lys Ala Arg Gly Ala Pro ValLeu 260 265 270 gag aaa aca ttt ggc tac cac ata cag tac ttt gca gag aacagc act 1103 Glu Lys Thr Phe Gly Tyr His Ile Gln Tyr Phe Ala Glu Asn SerThr 275 280 285 aac ctc aca gag ata aac aac atc acc acc cag cag tat gaactg ctt 1151 Asn Leu Thr Glu Ile Asn Asn Ile Thr Thr Gln Gln Tyr Glu LeuLeu 290 295 300 305 ctg atg agc cag gca cac tct gtg tcc gtg act tct tttaat tct ctt 1199 Leu Met Ser Gln Ala His Ser Val Ser Val Thr Ser Phe AsnSer Leu 310 315 320 ggc aag tcc caa gag acc atc ctg agg atc cca gat gtccat gag aag 1247 Gly Lys Ser Gln Glu Thr Ile Leu Arg Ile Pro Asp Val HisGlu Lys 325 330 335 acc ttc cag tac att aag agc atg cag gcc tac ata gccgag ccc ctg 1295 Thr Phe Gln Tyr Ile Lys Ser Met Gln Ala Tyr Ile Ala GluPro Leu 340 345 350 ttg gtg gtg aac tgg caa agc tcc att cct gcg gtg gacact tgg ata 1343 Leu Val Val Asn Trp Gln Ser Ser Ile Pro Ala Val Asp ThrTrp Ile 355 360 365 gtg gag tgg ctc cca gaa gct gcc atg tcg aag ttc cctgcc ctt tcc 1391 Val Glu Trp Leu Pro Glu Ala Ala Met Ser Lys Phe Pro AlaLeu Ser 370 375 380 385 tgg gaa tct gtg tct cag gtc acg aac tgg acc atcgag caa gat aaa 1439 Trp Glu Ser Val Ser Gln Val Thr Asn Trp Thr Ile GluGln Asp Lys 390 395 400 cta aaa cct ttc aca tgc tat aat ata tca gtg tatcca gtg ttg gga 1487 Leu Lys Pro Phe Thr Cys Tyr Asn Ile Ser Val Tyr ProVal Leu Gly 405 410 415 cac cga gtt gga gag ccg tat tca atc caa gct tatgcc aaa gaa gga 1535 His Arg Val Gly Glu Pro Tyr Ser Ile Gln Ala Tyr AlaLys Glu Gly 420 425 430 act cca tta aaa ggt cct gag acc agg gtg gag aacatc ggt ctg agg 1583 Thr Pro Leu Lys Gly Pro Glu Thr Arg Val Glu Asn IleGly Leu Arg 435 440 445 aca gcc acg atc aca tgg aag gag att cct aag agtgct agg aat gga 1631 Thr Ala Thr Ile Thr Trp Lys Glu Ile Pro Lys Ser AlaArg Asn Gly 450 455 460 465 ttt atc aac aat tac act gta ttt tac caa gctgaa ggt gga aaa gaa 1679 Phe Ile Asn Asn Tyr Thr Val Phe Tyr Gln Ala GluGly Gly Lys Glu 470 475 480 ctc tcc aag act gtt aac tct cat gcc ctg cagtgt gac ctg gag tct 1727 Leu Ser Lys Thr Val Asn Ser His Ala Leu Gln CysAsp Leu Glu Ser 485 490 495 ctg aca cga agg acc tct tat act gtt tgg gtcatg gcc agc acc aga 1775 Leu Thr Arg Arg Thr Ser Tyr Thr Val Trp Val MetAla Ser Thr Arg 500 505 510 gct gga ggt acc aac ggg gtg aga ata aac ttcaag aca ttg tca atc 1823 Ala Gly Gly Thr Asn Gly Val Arg Ile Asn Phe LysThr Leu Ser Ile 515 520 525 agt gtg ttt gaa att gtc ctt cta aca tct ctagtt gga gga ggc ctt 1871 Ser Val Phe Glu Ile Val Leu Leu Thr Ser Leu ValGly Gly Gly Leu 530 535 540 545 ctt cta ctt agc atc aaa aca gtg act tttggc ctc aga aag cca aac 1919 Leu Leu Leu Ser Ile Lys Thr Val Thr Phe GlyLeu Arg Lys Pro Asn 550 555 560 cgg ttg act ccc ctg tgt tgt cct gat gttccc aac cct gct gaa agt 1967 Arg Leu Thr Pro Leu Cys Cys Pro Asp Val ProAsn Pro Ala Glu Ser 565 570 575 agt tta gcc aca tgg ctc gga gat ggt ttcaag aag tca aat atg aag 2015 Ser Leu Ala Thr Trp Leu Gly Asp Gly Phe LysLys Ser Asn Met Lys 580 585 590 gag act gga aac tct ggg aac aca gaa gacgtg gtc cta aaa cca tgt 2063 Glu Thr Gly Asn Ser Gly Asn Thr Glu Asp ValVal Leu Lys Pro Cys 595 600 605 ccc gtc ccc gcg gat ctc att gac aag ctggta gtg aac ttt gag aat 2111 Pro Val Pro Ala Asp Leu Ile Asp Lys Leu ValVal Asn Phe Glu Asn 610 615 620 625 ttt ctg gaa gta gtt ttg aca gag gaagct gga aag ggt cag gcg agc 2159 Phe Leu Glu Val Val Leu Thr Glu Glu AlaGly Lys Gly Gln Ala Ser 630 635 640 att ttg gga gga gaa gcg aat gag tatatc tta tcc cag gaa cca agc 2207 Ile Leu Gly Gly Glu Ala Asn Glu Tyr IleLeu Ser Gln Glu Pro Ser 645 650 655 tgt cct ggc cat tgc tgaagctaccctcagggtcc aggacagctg tcttgttggc 2262 Cys Pro Gly His Cys 660 acttgactctggcaggaacc tgatctctac ttttcttctc cctgtctccg gacactttct 2322 ctccttcatgcagagaccag gactagagcg gattcctcat ggtttgccag gctcctcagt 2382 ccttgctcgggctcaggatc ttcaacaatg ccctttctgg gacactccat catccactta 2442 tatttattttttgcaacatt gtggattgaa cccagggact tgtttatgcg cgcaacttca 2502 gtaactgtggcagagactta ggaatggaga tctgaccctt tgcagaaggt ttctggacat 2562 ccgtccctgtgtgagcctca gacagcattg tctttacttt gaatcagctt ccaagttaat 2622 aaaagaaaaacagagaggtg gcataacagc tcctgcttcc tgacctgctt gagttccagt 2682 tctgacttcctttggtgatg aacagcaatg tgggaagtgt aagctgaata aaccctttcc 2742 tcccca 274857 662 PRT mus musculus 57 Met Leu Ser Ser Gln Lys Gly Ser Cys Ser GlnGlu Pro Gly Ala Ala 1 5 10 15 His Val Gln Pro Leu Gly Val Asn Ala GlyIle Met Trp Thr Leu Ala 20 25 30 Leu Trp Ala Phe Ser Phe Leu Cys Lys PheSer Leu Ala Val Leu Pro 35 40 45 Thr Lys Pro Glu Asn Ile Ser Cys Val PheTyr Phe Asp Arg Asn Leu 50 55 60 Thr Cys Thr Trp Arg Pro Glu Lys Glu ThrAsn Asp Thr Ser Tyr Ile 65 70 75 80 Val Thr Leu Thr Tyr Ser Tyr Gly LysSer Asn Tyr Ser Asp Asn Ala 85 90 95 Thr Glu Ala Ser Tyr Ser Phe Pro ArgSer Cys Ala Met Pro Pro Asp 100 105 110 Ile Cys Ser Val Glu Val Gln AlaGln Asn Gly Asp Gly Lys Val Lys 115 120 125 Ser Asp Ile Thr Tyr Trp HisLeu Ile Ser Ile Ala Lys Thr Glu Pro 130 135 140 Pro Ile Ile Leu Ser ValAsn Pro Ile Cys Asn Arg Met Phe Gln Ile 145 150 155 160 Gln Trp Lys ProArg Glu Lys Thr Arg Gly Phe Pro Leu Val Cys Met 165 170 175 Leu Arg PheArg Thr Val Asn Ser Ser Arg Trp Thr Glu Val Asn Phe 180 185 190 Glu AsnCys Lys Gln Val Cys Asn Leu Thr Gly Leu Gln Ala Phe Thr 195 200 205 GluTyr Val Leu Ala Leu Arg Phe Arg Phe Asn Asp Ser Arg Tyr Trp 210 215 220Ser Lys Trp Ser Lys Glu Glu Thr Arg Val Thr Met Glu Glu Val Pro 225 230235 240 His Val Leu Asp Leu Trp Arg Ile Leu Glu Pro Ala Asp Met Asn Gly245 250 255 Asp Arg Lys Val Arg Leu Leu Trp Lys Lys Ala Arg Gly Ala ProVal 260 265 270 Leu Glu Lys Thr Phe Gly Tyr His Ile Gln Tyr Phe Ala GluAsn Ser 275 280 285 Thr Asn Leu Thr Glu Ile Asn Asn Ile Thr Thr Gln GlnTyr Glu Leu 290 295 300 Leu Leu Met Ser Gln Ala His Ser Val Ser Val ThrSer Phe Asn Ser 305 310 315 320 Leu Gly Lys Ser Gln Glu Thr Ile Leu ArgIle Pro Asp Val His Glu 325 330 335 Lys Thr Phe Gln Tyr Ile Lys Ser MetGln Ala Tyr Ile Ala Glu Pro 340 345 350 Leu Leu Val Val Asn Trp Gln SerSer Ile Pro Ala Val Asp Thr Trp 355 360 365 Ile Val Glu Trp Leu Pro GluAla Ala Met Ser Lys Phe Pro Ala Leu 370 375 380 Ser Trp Glu Ser Val SerGln Val Thr Asn Trp Thr Ile Glu Gln Asp 385 390 395 400 Lys Leu Lys ProPhe Thr Cys Tyr Asn Ile Ser Val Tyr Pro Val Leu 405 410 415 Gly His ArgVal Gly Glu Pro Tyr Ser Ile Gln Ala Tyr Ala Lys Glu 420 425 430 Gly ThrPro Leu Lys Gly Pro Glu Thr Arg Val Glu Asn Ile Gly Leu 435 440 445 ArgThr Ala Thr Ile Thr Trp Lys Glu Ile Pro Lys Ser Ala Arg Asn 450 455 460Gly Phe Ile Asn Asn Tyr Thr Val Phe Tyr Gln Ala Glu Gly Gly Lys 465 470475 480 Glu Leu Ser Lys Thr Val Asn Ser His Ala Leu Gln Cys Asp Leu Glu485 490 495 Ser Leu Thr Arg Arg Thr Ser Tyr Thr Val Trp Val Met Ala SerThr 500 505 510 Arg Ala Gly Gly Thr Asn Gly Val Arg Ile Asn Phe Lys ThrLeu Ser 515 520 525 Ile Ser Val Phe Glu Ile Val Leu Leu Thr Ser Leu ValGly Gly Gly 530 535 540 Leu Leu Leu Leu Ser Ile Lys Thr Val Thr Phe GlyLeu Arg Lys Pro 545 550 555 560 Asn Arg Leu Thr Pro Leu Cys Cys Pro AspVal Pro Asn Pro Ala Glu 565 570 575 Ser Ser Leu Ala Thr Trp Leu Gly AspGly Phe Lys Lys Ser Asn Met 580 585 590 Lys Glu Thr Gly Asn Ser Gly AsnThr Glu Asp Val Val Leu Lys Pro 595 600 605 Cys Pro Val Pro Ala Asp LeuIle Asp Lys Leu Val Val Asn Phe Glu 610 615 620 Asn Phe Leu Glu Val ValLeu Thr Glu Glu Ala Gly Lys Gly Gln Ala 625 630 635 640 Ser Ile Leu GlyGly Glu Ala Asn Glu Tyr Ile Leu Ser Gln Glu Pro 645 650 655 Ser Cys ProGly His Cys 660 58 21 DNA Artificial Sequence Oligonucleotide primerZC6673 58 gcgcaaggtg ccgttcacag c 21 59 36 DNA Artificial SequenceOligonucleotide primer ZC29082 59 caatttgttg ggttttttta gcagcagtaggcccag 36 60 36 DNA Artificial Sequence Oligonucleotide primer ZC2908360 ctgggcctac tgctgctaaa aaaacccaac aaattg 36 61 36 DNA ArtificialSequence Oligonucleotide primer ZC29145 61 gcgtctagag ggttatattgaagttgggca ggaaga 36 62 33 DNA Artificial Sequence Oligonucleotideprimer ZC29359 62 gcgggatcca tgaagctctc tccccagcct tca 33 63 23 DNAArtificial Sequence Oligonucleotide primer ZC27899 63 ccagaactttgactccttga ccg 23 64 23 DNA Artificial Sequence Oligonucleotide primerZC27895 64 gaagtcaact tcgctaagaa ccg 23 65 34 DNA Artificial SequenceOligonucleotide primer ZC29122 65 ccgctcgagt tatattgaag ttgggcagga agac34 66 33 DNA Artificial Sequence Oligonucleotide primer ZC29451 66ccggaattcc cctgatacat gaagctctct ccc 33 67 33 DNA Artificial SequenceOligonucleotide primer ZC29124 67 cgcggatccc tcaaagacac tgaatgacaa tgt33 68 2295 DNA Artificial Sequence Polynucleitide encoding humanzcytor17-Fc4 fusion 68 atg aag ctc tct ccc cag cct tca tgt gtt aac ctgggg atg atg tgg 48 Met Lys Leu Ser Pro Gln Pro Ser Cys Val Asn Leu GlyMet Met Trp 1 5 10 15 acc tgg gca ctg tgg atg ctc cct tca ctc tgc aaattc agc ctg gca 96 Thr Trp Ala Leu Trp Met Leu Pro Ser Leu Cys Lys PheSer Leu Ala 20 25 30 gct ctg cca gct aag cct gag aac att tcc tgt gtc tactac tat agg 144 Ala Leu Pro Ala Lys Pro Glu Asn Ile Ser Cys Val Tyr TyrTyr Arg 35 40 45 aaa aat tta acc tgc act tgg agt cca gga aag gaa acc agttat acc 192 Lys Asn Leu Thr Cys Thr Trp Ser Pro Gly Lys Glu Thr Ser TyrThr 50 55 60 cag tac aca gtt aag aga act tac gct ttt gga gaa aaa cat gataat 240 Gln Tyr Thr Val Lys Arg Thr Tyr Ala Phe Gly Glu Lys His Asp Asn65 70 75 80 tgt aca acc aat agt tct aca agt gaa aat cgt gct tcg tgc tctttt 288 Cys Thr Thr Asn Ser Ser Thr Ser Glu Asn Arg Ala Ser Cys Ser Phe85 90 95 ttc ctt cca aga ata acg atc cca gat aat tat acc att gag gtg gaa336 Phe Leu Pro Arg Ile Thr Ile Pro Asp Asn Tyr Thr Ile Glu Val Glu 100105 110 gct gaa aat gga gat ggt gta att aaa tct cat atg aca tac tgg aga384 Ala Glu Asn Gly Asp Gly Val Ile Lys Ser His Met Thr Tyr Trp Arg 115120 125 tta gag aac ata gcg aaa act gaa cca cct aag att ttc cgt gtg aaa432 Leu Glu Asn Ile Ala Lys Thr Glu Pro Pro Lys Ile Phe Arg Val Lys 130135 140 cca gtt ttg ggc atc aaa cga atg att caa att gaa tgg ata aag cct480 Pro Val Leu Gly Ile Lys Arg Met Ile Gln Ile Glu Trp Ile Lys Pro 145150 155 160 gag ttg gcg cct gtt tca tct gat tta aaa tac aca ctt cga ttcagg 528 Glu Leu Ala Pro Val Ser Ser Asp Leu Lys Tyr Thr Leu Arg Phe Arg165 170 175 aca gtc aac agt acc agc tgg atg gaa gtc aac ttc gct aag aaccgt 576 Thr Val Asn Ser Thr Ser Trp Met Glu Val Asn Phe Ala Lys Asn Arg180 185 190 aag gat aaa aac caa acg tac aac ctc acg ggg ctg cag cct tttaca 624 Lys Asp Lys Asn Gln Thr Tyr Asn Leu Thr Gly Leu Gln Pro Phe Thr195 200 205 gaa tat gtc ata gct ctg cga tgt gcg gtc aag gag tca aag ttctgg 672 Glu Tyr Val Ile Ala Leu Arg Cys Ala Val Lys Glu Ser Lys Phe Trp210 215 220 agt gac tgg agc caa gaa aaa atg gga atg act gag gaa gaa gctcca 720 Ser Asp Trp Ser Gln Glu Lys Met Gly Met Thr Glu Glu Glu Ala Pro225 230 235 240 tgt ggc ctg gaa ctg tgg aga gtc ctg aaa cca gct gag gcggat gga 768 Cys Gly Leu Glu Leu Trp Arg Val Leu Lys Pro Ala Glu Ala AspGly 245 250 255 aga agg cca gtg cgg ttg tta tgg aag aag gca aga gga gcccca gtc 816 Arg Arg Pro Val Arg Leu Leu Trp Lys Lys Ala Arg Gly Ala ProVal 260 265 270 cta gag aaa aca ctt ggc tac aac ata tgg tac tat cca gaaagc aac 864 Leu Glu Lys Thr Leu Gly Tyr Asn Ile Trp Tyr Tyr Pro Glu SerAsn 275 280 285 act aac ctc aca gaa aca atg aac act act aac cag cag cttgaa ctg 912 Thr Asn Leu Thr Glu Thr Met Asn Thr Thr Asn Gln Gln Leu GluLeu 290 295 300 cat ctg gga ggc gag agc ttt tgg gtg tct atg att tct tataat tct 960 His Leu Gly Gly Glu Ser Phe Trp Val Ser Met Ile Ser Tyr AsnSer 305 310 315 320 ctt ggg aag tct cca gtg gcc acc ctg agg att cca gctatt caa gaa 1008 Leu Gly Lys Ser Pro Val Ala Thr Leu Arg Ile Pro Ala IleGln Glu 325 330 335 aaa tca ttt cag tgc att gag gtc atg cag gcc tgc gttgct gag gac 1056 Lys Ser Phe Gln Cys Ile Glu Val Met Gln Ala Cys Val AlaGlu Asp 340 345 350 cag cta gtg gtg aag tgg caa agc tct gct cta gac gtgaac act tgg 1104 Gln Leu Val Val Lys Trp Gln Ser Ser Ala Leu Asp Val AsnThr Trp 355 360 365 atg att gaa tgg ttt ccg gat gtg gac tca gag ccc accacc ctt tcc 1152 Met Ile Glu Trp Phe Pro Asp Val Asp Ser Glu Pro Thr ThrLeu Ser 370 375 380 tgg gaa tct gtg tct cag gcc acg aac tgg acg atc cagcaa gat aaa 1200 Trp Glu Ser Val Ser Gln Ala Thr Asn Trp Thr Ile Gln GlnAsp Lys 385 390 395 400 tta aaa ccc ttc tgg tgc tat aac atc tct gtg tatcca atg ttg cat 1248 Leu Lys Pro Phe Trp Cys Tyr Asn Ile Ser Val Tyr ProMet Leu His 405 410 415 gac aaa gtt ggc gag cca tat tcc atc cag gct tatgcc aaa gaa ggc 1296 Asp Lys Val Gly Glu Pro Tyr Ser Ile Gln Ala Tyr AlaLys Glu Gly 420 425 430 gtt cca tca gaa ggt cct gag acc aag gtg gag aacatt ggc gtg aag 1344 Val Pro Ser Glu Gly Pro Glu Thr Lys Val Glu Asn IleGly Val Lys 435 440 445 acg gtc acg atc aca tgg aaa gag att ccc aag agtgag aga aag ggt 1392 Thr Val Thr Ile Thr Trp Lys Glu Ile Pro Lys Ser GluArg Lys Gly 450 455 460 atc atc tgc aac tac acc atc ttt tac caa gct gaaggt gga aaa gga 1440 Ile Ile Cys Asn Tyr Thr Ile Phe Tyr Gln Ala Glu GlyGly Lys Gly 465 470 475 480 ttc tcc aag aca gtc aat tcc agc atc ttg cagtac ggc ctg gag tcc 1488 Phe Ser Lys Thr Val Asn Ser Ser Ile Leu Gln TyrGly Leu Glu Ser 485 490 495 ctg aaa cga aag acc tct tac att gtt cag gtcatg gcc agc acc agt 1536 Leu Lys Arg Lys Thr Ser Tyr Ile Val Gln Val MetAla Ser Thr Ser 500 505 510 gct ggg gga acc aac ggg acc agc ata aat ttcaag aca ttg tca ttc 1584 Ala Gly Gly Thr Asn Gly Thr Ser Ile Asn Phe LysThr Leu Ser Phe 515 520 525 agt gtc ttt gag gag ccc aga tct tca gac aaaact cac aca tgc cca 1632 Ser Val Phe Glu Glu Pro Arg Ser Ser Asp Lys ThrHis Thr Cys Pro 530 535 540 ccg tgc cca gca cct gaa gcc gag ggg gca ccgtca gtc ttc ctc ttc 1680 Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro SerVal Phe Leu Phe 545 550 555 560 ccc cca aaa ccc aag gac acc ctc atg atctcc cgg acc cct gag gtc 1728 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile SerArg Thr Pro Glu Val 565 570 575 aca tgc gtg gtg gtg gac gtg agc cac gaagac cct gag gtc aag ttc 1776 Thr Cys Val Val Val Asp Val Ser His Glu AspPro Glu Val Lys Phe 580 585 590 aac tgg tac gtg gac ggc gtg gag gtg cataat gcc aag aca aag ccg 1824 Asn Trp Tyr Val Asp Gly Val Glu Val His AsnAla Lys Thr Lys Pro 595 600 605 cgg gag gag cag tac aac agc acg tac cgtgtg gtc agc gtc ctc acc 1872 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg ValVal Ser Val Leu Thr 610 615 620 gtc ctg cac cag gac tgg ctg aat ggc aaggag tac aag tgc aag gtc 1920 Val Leu His Gln Asp Trp Leu Asn Gly Lys GluTyr Lys Cys Lys Val 625 630 635 640 tcc aac aaa gcc ctc cca tcc tcc atcgag aaa acc atc tcc aaa gcc 1968 Ser Asn Lys Ala Leu Pro Ser Ser Ile GluLys Thr Ile Ser Lys Ala 645 650 655 aaa ggg cag ccc cga gaa cca cag gtgtac acc ctg ccc cca tcc cgg 2016 Lys Gly Gln Pro Arg Glu Pro Gln Val TyrThr Leu Pro Pro Ser Arg 660 665 670 gat gag ctg acc aag aac cag gtc agcctg acc tgc ctg gtc aaa ggc 2064 Asp Glu Leu Thr Lys Asn Gln Val Ser LeuThr Cys Leu Val Lys Gly 675 680 685 ttc tat ccc agc gac atc gcc gtg gagtgg gag agc aat ggg cag ccg 2112 Phe Tyr Pro Ser Asp Ile Ala Val Glu TrpGlu Ser Asn Gly Gln Pro 690 695 700 gag aac aac tac aag acc acg cct cccgtg ctg gac tcc gac ggc tcc 2160 Glu Asn Asn Tyr Lys Thr Thr Pro Pro ValLeu Asp Ser Asp Gly Ser 705 710 715 720 ttc ttc ctc tac agc aag ctc accgtg gac aag agc agg tgg cag cag 2208 Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln 725 730 735 ggg aac gtc ttc tca tgc tcc gtgatg cat gag gct ctg cac aac cac 2256 Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His Asn His 740 745 750 tac acg cag aag agc ctc tcc ctgtct ccg ggt aaa taa 2295 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlyLys * 755 760 69 764 PRT Artificial Sequence Human Zcytor17-Fc4 fusionpolypeptide 69 Met Lys Leu Ser Pro Gln Pro Ser Cys Val Asn Leu Gly MetMet Trp 1 5 10 15 Thr Trp Ala Leu Trp Met Leu Pro Ser Leu Cys Lys PheSer Leu Ala 20 25 30 Ala Leu Pro Ala Lys Pro Glu Asn Ile Ser Cys Val TyrTyr Tyr Arg 35 40 45 Lys Asn Leu Thr Cys Thr Trp Ser Pro Gly Lys Glu ThrSer Tyr Thr 50 55 60 Gln Tyr Thr Val Lys Arg Thr Tyr Ala Phe Gly Glu LysHis Asp Asn 65 70 75 80 Cys Thr Thr Asn Ser Ser Thr Ser Glu Asn Arg AlaSer Cys Ser Phe 85 90 95 Phe Leu Pro Arg Ile Thr Ile Pro Asp Asn Tyr ThrIle Glu Val Glu 100 105 110 Ala Glu Asn Gly Asp Gly Val Ile Lys Ser HisMet Thr Tyr Trp Arg 115 120 125 Leu Glu Asn Ile Ala Lys Thr Glu Pro ProLys Ile Phe Arg Val Lys 130 135 140 Pro Val Leu Gly Ile Lys Arg Met IleGln Ile Glu Trp Ile Lys Pro 145 150 155 160 Glu Leu Ala Pro Val Ser SerAsp Leu Lys Tyr Thr Leu Arg Phe Arg 165 170 175 Thr Val Asn Ser Thr SerTrp Met Glu Val Asn Phe Ala Lys Asn Arg 180 185 190 Lys Asp Lys Asn GlnThr Tyr Asn Leu Thr Gly Leu Gln Pro Phe Thr 195 200 205 Glu Tyr Val IleAla Leu Arg Cys Ala Val Lys Glu Ser Lys Phe Trp 210 215 220 Ser Asp TrpSer Gln Glu Lys Met Gly Met Thr Glu Glu Glu Ala Pro 225 230 235 240 CysGly Leu Glu Leu Trp Arg Val Leu Lys Pro Ala Glu Ala Asp Gly 245 250 255Arg Arg Pro Val Arg Leu Leu Trp Lys Lys Ala Arg Gly Ala Pro Val 260 265270 Leu Glu Lys Thr Leu Gly Tyr Asn Ile Trp Tyr Tyr Pro Glu Ser Asn 275280 285 Thr Asn Leu Thr Glu Thr Met Asn Thr Thr Asn Gln Gln Leu Glu Leu290 295 300 His Leu Gly Gly Glu Ser Phe Trp Val Ser Met Ile Ser Tyr AsnSer 305 310 315 320 Leu Gly Lys Ser Pro Val Ala Thr Leu Arg Ile Pro AlaIle Gln Glu 325 330 335 Lys Ser Phe Gln Cys Ile Glu Val Met Gln Ala CysVal Ala Glu Asp 340 345 350 Gln Leu Val Val Lys Trp Gln Ser Ser Ala LeuAsp Val Asn Thr Trp 355 360 365 Met Ile Glu Trp Phe Pro Asp Val Asp SerGlu Pro Thr Thr Leu Ser 370 375 380 Trp Glu Ser Val Ser Gln Ala Thr AsnTrp Thr Ile Gln Gln Asp Lys 385 390 395 400 Leu Lys Pro Phe Trp Cys TyrAsn Ile Ser Val Tyr Pro Met Leu His 405 410 415 Asp Lys Val Gly Glu ProTyr Ser Ile Gln Ala Tyr Ala Lys Glu Gly 420 425 430 Val Pro Ser Glu GlyPro Glu Thr Lys Val Glu Asn Ile Gly Val Lys 435 440 445 Thr Val Thr IleThr Trp Lys Glu Ile Pro Lys Ser Glu Arg Lys Gly 450 455 460 Ile Ile CysAsn Tyr Thr Ile Phe Tyr Gln Ala Glu Gly Gly Lys Gly 465 470 475 480 PheSer Lys Thr Val Asn Ser Ser Ile Leu Gln Tyr Gly Leu Glu Ser 485 490 495Leu Lys Arg Lys Thr Ser Tyr Ile Val Gln Val Met Ala Ser Thr Ser 500 505510 Ala Gly Gly Thr Asn Gly Thr Ser Ile Asn Phe Lys Thr Leu Ser Phe 515520 525 Ser Val Phe Glu Glu Pro Arg Ser Ser Asp Lys Thr His Thr Cys Pro530 535 540 Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe LeuPhe 545 550 555 560 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg ThrPro Glu Val 565 570 575 Thr Cys Val Val Val Asp Val Ser His Glu Asp ProGlu Val Lys Phe 580 585 590 Asn Trp Tyr Val Asp Gly Val Glu Val His AsnAla Lys Thr Lys Pro 595 600 605 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr ArgVal Val Ser Val Leu Thr 610 615 620 Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val 625 630 635 640 Ser Asn Lys Ala Leu Pro SerSer Ile Glu Lys Thr Ile Ser Lys Ala 645 650 655 Lys Gly Gln Pro Arg GluPro Gln Val Tyr Thr Leu Pro Pro Ser Arg 660 665 670 Asp Glu Leu Thr LysAsn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 675 680 685 Phe Tyr Pro SerAsp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 690 695 700 Glu Asn AsnTyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 705 710 715 720 PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 725 730 735Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 740 745750 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 755 760 70 34 DNAArtificial Sequence Oligonucleotide primer ZC29157 70 ctagtatggccggccatgaa gctctctccc cagc 34 71 41 DNA Artificial SequenceOligonucleotide primer ZC29150 71 gtctgaagat ctgggctcct caaagacactgaatgacaat g 41 72 22 DNA Artificial Sequence Oligonucleotide primerZC29180 72 cctggagtcc ctgaaacgaa ag 22 73 20 DNA Artificial SequenceOligonucleotide primer ZC28917 73 tgcaagatgc tggaattgac 20 74 19 DNAArtificial Sequence Oligonucleotide primer ZC29179 74 gcagggttgggaacggtgg 19 75 20 DNA Artificial Sequence Oligonucleotide primerZC28916 75 agtcaattcc agcatcttgc 20 76 20 DNA Artificial SequenceOligonucleotide primer ZC28918 76 tcacagagtc atcagactcc 20 77 21 DNAArtificial Sequence Oligonucleotide primer ZC38065 77 ctttcctgggaatctgtgtc t 21 78 18 DNA Artificial Sequence Oligonucleotide primerZC38068 78 cctccagctc tggtgctg 18 79 20 DNA Artificial SequenceOligonucleotide primer ZC10651 79 agcttttctg cagcagctct 20 80 23 DNAArtificial Sequence Oligonucleotide primer ZC10565 80 tttgcagaaaaggttgcaaa tgc 23 81 24 DNA Artificial Sequence Oligonucleotide primerZC37877 81 caaaaaaccc aacaaattga ctca 24 82 25 DNA Artificial SequenceOligonucleotide primer ZC37876 82 catgtggcta tactactttc agcag 25 83 22DNA Artificial Sequence Oligonucleotide primer ZC37776 zcytor17TaqMan(r) probe 83 ctgtgttggc ccaccgttcc ca 22 84 20 DNA ArtificialSequence rRNA forward primer 84 cggctaccac atccaaggaa 20 85 18 DNAArtificial Sequence rRNA reverse primer 85 gctggaatta ccgcggct 18 86 22DNA Artificial Sequence rRNA TaqMan(r) probe 86 tgctggcacc agacttgccc tc22 87 21 DNA Artificial Sequence Oligonucleotide primer ZC22276 87gcttgccctt cagcatgtag a 21 88 19 DNA Artificial Sequence Oligonucleotideprimer ZC38239 88 gccgactaag ccagagaac 19 89 20 DNA Artificial SequenceOligonucleotide primer ZC38245 89 ctgttgacag ttctgaaccg 20 90 20 DNAArtificial Sequence Oligonucleotide primer ZC38238 90 cgcggtttccattgtatctg 20 91 8 PRT Artificial Sequence Modified Glu-Glu tag peptide91 Gly Ser Glu Tyr Met Pro Met Glu 1 5 92 2728 DNA Mus musculus CDS(237)...(1877) 92 gatggggccc tgaatgttga tctgacagaa ttccagacca acctggtggttattgtcctt 60 ttcatctggt catgctgaat atactctcaa gatgtgctgg agaaggtgctgctgtccggg 120 ctctcagaga aggcagtgct ggaggcgttc ctggcccggg tctcctcctactgttcctgg 180 tagcccagcc ttctcggggt ggaaggagaa gctggccagg tgagctctgaggaagc atg 239 Met 1 ctg agc agc cag aag gga tcc tgc agc cag gaa cca ggggca gcc cac 287 Leu Ser Ser Gln Lys Gly Ser Cys Ser Gln Glu Pro Gly AlaAla His 5 10 15 gtc cag cct ctg ggt gtg aac gct gga ata atg tgg acc ttggca ctg 335 Val Gln Pro Leu Gly Val Asn Ala Gly Ile Met Trp Thr Leu AlaLeu 20 25 30 tgg gca ttc tct ttc ctc tgc aaa ttc agc ctg gca gtc ctg ccgact 383 Trp Ala Phe Ser Phe Leu Cys Lys Phe Ser Leu Ala Val Leu Pro Thr35 40 45 aag cca gag aac att tcc tgc gtc ttt tac ttc gac aga aat ctg act431 Lys Pro Glu Asn Ile Ser Cys Val Phe Tyr Phe Asp Arg Asn Leu Thr 5055 60 65 tgc act tgg aga cca gag aag gaa acc aat gat acc agc tac att gtg479 Cys Thr Trp Arg Pro Glu Lys Glu Thr Asn Asp Thr Ser Tyr Ile Val 7075 80 act ttg act tac tcc tat gga aaa agc aat tat agt gac aat gct aca527 Thr Leu Thr Tyr Ser Tyr Gly Lys Ser Asn Tyr Ser Asp Asn Ala Thr 8590 95 gag gct tca tat tct ttt ccc cgt tcc tgt gca atg ccc cca gac atc575 Glu Ala Ser Tyr Ser Phe Pro Arg Ser Cys Ala Met Pro Pro Asp Ile 100105 110 tgc agt gtt gaa gta caa gct caa aat gga gat ggt aaa gtt aaa tct623 Cys Ser Val Glu Val Gln Ala Gln Asn Gly Asp Gly Lys Val Lys Ser 115120 125 gac atc aca tat tgg cat tta atc tcc ata gca aaa acc gaa cca cct671 Asp Ile Thr Tyr Trp His Leu Ile Ser Ile Ala Lys Thr Glu Pro Pro 130135 140 145 ata att tta agt gtg aat cca att tgt aat aga atg ttc cag atacaa 719 Ile Ile Leu Ser Val Asn Pro Ile Cys Asn Arg Met Phe Gln Ile Gln150 155 160 tgg aaa ccg cgt gaa aag act cgt ggg ttt cct tta gta tgc atgctt 767 Trp Lys Pro Arg Glu Lys Thr Arg Gly Phe Pro Leu Val Cys Met Leu165 170 175 cgg ttc aga act gtc aac agt agc cgc tgg acg gaa gtc aat tttgaa 815 Arg Phe Arg Thr Val Asn Ser Ser Arg Trp Thr Glu Val Asn Phe Glu180 185 190 aac tgt aaa cag gtc tgc aac ctc aca gga ctt cag gct ttc acagaa 863 Asn Cys Lys Gln Val Cys Asn Leu Thr Gly Leu Gln Ala Phe Thr Glu195 200 205 tat gtc ctg gct cta cga ttc agg ttc aat gac tca aga tat tggagc 911 Tyr Val Leu Ala Leu Arg Phe Arg Phe Asn Asp Ser Arg Tyr Trp Ser210 215 220 225 aag tgg agc aaa gaa gaa acc aga gtg act atg gag gaa gttcca cat 959 Lys Trp Ser Lys Glu Glu Thr Arg Val Thr Met Glu Glu Val ProHis 230 235 240 gtc ctg gac ctg tgg aga att ctg gaa cca gca gac atg aacgga gac 1007 Val Leu Asp Leu Trp Arg Ile Leu Glu Pro Ala Asp Met Asn GlyAsp 245 250 255 agg aag gtg cga ttg ctg tgg aag aag gca aga gga gcc cccgtc ttg 1055 Arg Lys Val Arg Leu Leu Trp Lys Lys Ala Arg Gly Ala Pro ValLeu 260 265 270 gag aaa aca ttt ggc tac cac ata cag tac ttt gca gag aacagc act 1103 Glu Lys Thr Phe Gly Tyr His Ile Gln Tyr Phe Ala Glu Asn SerThr 275 280 285 aac ctc aca gag ata aac aac atc acc acc cag cag tat gaactg ctt 1151 Asn Leu Thr Glu Ile Asn Asn Ile Thr Thr Gln Gln Tyr Glu LeuLeu 290 295 300 305 ctg atg agc cag gca cac tct gtg tcc gtg act tct tttaat tct ctt 1199 Leu Met Ser Gln Ala His Ser Val Ser Val Thr Ser Phe AsnSer Leu 310 315 320 ggc aag tcc caa gag acc atc ctg agg atc cca gat gtccat gag aag 1247 Gly Lys Ser Gln Glu Thr Ile Leu Arg Ile Pro Asp Val HisGlu Lys 325 330 335 acc ttc cag tac att aag agc atg cag gcc tac ata gccgag ccc ctg 1295 Thr Phe Gln Tyr Ile Lys Ser Met Gln Ala Tyr Ile Ala GluPro Leu 340 345 350 ttg gtg gtg aac tgg caa agc tcc att cct gcg gtg gacact tgg ata 1343 Leu Val Val Asn Trp Gln Ser Ser Ile Pro Ala Val Asp ThrTrp Ile 355 360 365 gtg gag tgg ctc cca gaa gct gcc atg tcg aag ttc cctgcc ctt tcc 1391 Val Glu Trp Leu Pro Glu Ala Ala Met Ser Lys Phe Pro AlaLeu Ser 370 375 380 385 tgg gaa tct gtg tct cag gtc acg aac tgg acc atcgag caa gat aaa 1439 Trp Glu Ser Val Ser Gln Val Thr Asn Trp Thr Ile GluGln Asp Lys 390 395 400 cta aaa cct ttc aca tgc tat aat ata tca gtg tatcca gtg ttg gga 1487 Leu Lys Pro Phe Thr Cys Tyr Asn Ile Ser Val Tyr ProVal Leu Gly 405 410 415 cac cga gtt gga gag ccg tat tca atc caa gct tatgcc aaa gaa gga 1535 His Arg Val Gly Glu Pro Tyr Ser Ile Gln Ala Tyr AlaLys Glu Gly 420 425 430 act cca tta aaa ggt cct gag acc agg gtg gag aacatc ggt ctg agg 1583 Thr Pro Leu Lys Gly Pro Glu Thr Arg Val Glu Asn IleGly Leu Arg 435 440 445 aca gcc acg atc aca tgg aag gag att cct aag agtgct agg aat gga 1631 Thr Ala Thr Ile Thr Trp Lys Glu Ile Pro Lys Ser AlaArg Asn Gly 450 455 460 465 ttt atc aac aat tac act gta ttt tac caa gctgaa ggt gga aaa gaa 1679 Phe Ile Asn Asn Tyr Thr Val Phe Tyr Gln Ala GluGly Gly Lys Glu 470 475 480 ctc tcc aag act gtt aac tct cat gcc ctg cagtgt gac ctg gag tct 1727 Leu Ser Lys Thr Val Asn Ser His Ala Leu Gln CysAsp Leu Glu Ser 485 490 495 ctg aca cga agg acc tct tat act gtt tgg gtcatg gcc agc acc aga 1775 Leu Thr Arg Arg Thr Ser Tyr Thr Val Trp Val MetAla Ser Thr Arg 500 505 510 gct gga ggt acc aac ggg gtg aga ata aac ttcaag aca ttg tca atc 1823 Ala Gly Gly Thr Asn Gly Val Arg Ile Asn Phe LysThr Leu Ser Ile 515 520 525 agt gag tac tgg ctt cag gcc tca ttc tgg agttta ctt cgg gtt gga 1871 Ser Glu Tyr Trp Leu Gln Ala Ser Phe Trp Ser LeuLeu Arg Val Gly 530 535 540 545 aat gtt tgacaggagc aaggagagcc agcagagggcagcagagcat ggcttctcct 1927 Asn Val gctctctctg gctcactcac ctcccaggagttactgagga gctggcaaag ggagggctga 1987 gttagaccaa caggccattt tgatccttgctggtaagcag ccacaaataa tcttaagatg 2047 aagcaagcaa catccacttc agcctcagccacgtcaaagg ctgttgcctg agctcacact 2107 ggccagttcc taaatgtcag gagttgtgcaatagaacctg ggaaggaaca actggttgat 2167 cagaggtcac tgacaaggga cttaatgttaccatctgcgg tggggctttt gtttcgtttt 2227 gtttgtttgt tatgtgtatt caacttatcagcttttacgt tgaaaacatg aaaagcaaga 2287 caaatttgtt agatatcaca tataatgtgaaatataatag tttaataatt gagtaggaaa 2347 gctgagggca tgtaatagac agagggaaaagaagaggaaa gccagtctgg tctacaaagt 2407 gagttccagg acagccaggg ctacatggagaaaccctgtc tcaatcaatc aatcaatcaa 2467 tcaatcagtc aatcaatcaa aattcaagcagcattgacaa gttttgcaat aactactata 2527 aaccaaaaaa gtcatcttga tgtatctcagaagccccttg ttatttatgt tcctgaagac 2587 taaagtagac cgtggctctg agaaccatgagcaagataac acgttctgtc ctgcagccta 2647 acaatgcctt cttggtattc tttttgatacaacttctaaa ataacttttt tttaaaaaaa 2707 ataaaaatca tgttacagct a 2728 93547 PRT Mus musculus 93 Met Leu Ser Ser Gln Lys Gly Ser Cys Ser Gln GluPro Gly Ala Ala 1 5 10 15 His Val Gln Pro Leu Gly Val Asn Ala Gly IleMet Trp Thr Leu Ala 20 25 30 Leu Trp Ala Phe Ser Phe Leu Cys Lys Phe SerLeu Ala Val Leu Pro 35 40 45 Thr Lys Pro Glu Asn Ile Ser Cys Val Phe TyrPhe Asp Arg Asn Leu 50 55 60 Thr Cys Thr Trp Arg Pro Glu Lys Glu Thr AsnAsp Thr Ser Tyr Ile 65 70 75 80 Val Thr Leu Thr Tyr Ser Tyr Gly Lys SerAsn Tyr Ser Asp Asn Ala 85 90 95 Thr Glu Ala Ser Tyr Ser Phe Pro Arg SerCys Ala Met Pro Pro Asp 100 105 110 Ile Cys Ser Val Glu Val Gln Ala GlnAsn Gly Asp Gly Lys Val Lys 115 120 125 Ser Asp Ile Thr Tyr Trp His LeuIle Ser Ile Ala Lys Thr Glu Pro 130 135 140 Pro Ile Ile Leu Ser Val AsnPro Ile Cys Asn Arg Met Phe Gln Ile 145 150 155 160 Gln Trp Lys Pro ArgGlu Lys Thr Arg Gly Phe Pro Leu Val Cys Met 165 170 175 Leu Arg Phe ArgThr Val Asn Ser Ser Arg Trp Thr Glu Val Asn Phe 180 185 190 Glu Asn CysLys Gln Val Cys Asn Leu Thr Gly Leu Gln Ala Phe Thr 195 200 205 Glu TyrVal Leu Ala Leu Arg Phe Arg Phe Asn Asp Ser Arg Tyr Trp 210 215 220 SerLys Trp Ser Lys Glu Glu Thr Arg Val Thr Met Glu Glu Val Pro 225 230 235240 His Val Leu Asp Leu Trp Arg Ile Leu Glu Pro Ala Asp Met Asn Gly 245250 255 Asp Arg Lys Val Arg Leu Leu Trp Lys Lys Ala Arg Gly Ala Pro Val260 265 270 Leu Glu Lys Thr Phe Gly Tyr His Ile Gln Tyr Phe Ala Glu AsnSer 275 280 285 Thr Asn Leu Thr Glu Ile Asn Asn Ile Thr Thr Gln Gln TyrGlu Leu 290 295 300 Leu Leu Met Ser Gln Ala His Ser Val Ser Val Thr SerPhe Asn Ser 305 310 315 320 Leu Gly Lys Ser Gln Glu Thr Ile Leu Arg IlePro Asp Val His Glu 325 330 335 Lys Thr Phe Gln Tyr Ile Lys Ser Met GlnAla Tyr Ile Ala Glu Pro 340 345 350 Leu Leu Val Val Asn Trp Gln Ser SerIle Pro Ala Val Asp Thr Trp 355 360 365 Ile Val Glu Trp Leu Pro Glu AlaAla Met Ser Lys Phe Pro Ala Leu 370 375 380 Ser Trp Glu Ser Val Ser GlnVal Thr Asn Trp Thr Ile Glu Gln Asp 385 390 395 400 Lys Leu Lys Pro PheThr Cys Tyr Asn Ile Ser Val Tyr Pro Val Leu 405 410 415 Gly His Arg ValGly Glu Pro Tyr Ser Ile Gln Ala Tyr Ala Lys Glu 420 425 430 Gly Thr ProLeu Lys Gly Pro Glu Thr Arg Val Glu Asn Ile Gly Leu 435 440 445 Arg ThrAla Thr Ile Thr Trp Lys Glu Ile Pro Lys Ser Ala Arg Asn 450 455 460 GlyPhe Ile Asn Asn Tyr Thr Val Phe Tyr Gln Ala Glu Gly Gly Lys 465 470 475480 Glu Leu Ser Lys Thr Val Asn Ser His Ala Leu Gln Cys Asp Leu Glu 485490 495 Ser Leu Thr Arg Arg Thr Ser Tyr Thr Val Trp Val Met Ala Ser Thr500 505 510 Arg Ala Gly Gly Thr Asn Gly Val Arg Ile Asn Phe Lys Thr LeuSer 515 520 525 Ile Ser Glu Tyr Trp Leu Gln Ala Ser Phe Trp Ser Leu LeuArg Val 530 535 540 Gly Asn Val 545

What is claimed is:
 1. An isolated polynucleotide, wherein thepolypeptide comprises a sequence of amino acid residues that is selectedfrom the group consisting of: (a) the amino acid sequence as shown inSEQ ID NO:2 from amino acid number 20 (Ala) to amino acid number 227(Pro); (b) the amino acid sequence as shown in SEQ ID NO:2 from aminoacid number 20 (Ala) to amino acid number 519 (Glu); (c) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Ala) toamino acid number 543 (Leu); (d) the amino acid sequence as shown in SEQID NO:2 from amino acid number 544 (Lys) to amino acid number 732 (Val);(e) the amino acid sequence as shown in SEQ ID NO:46 from amino acidnumber 544 (Lys) to amino acid number 649 (Ile); (f) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Ala) toamino acid number 732 (Val); (g) the amino acid sequence as shown in SEQID NO:46 from amino acid number 20 (Ala) to amino acid number 649 (Ile);(h) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 1 (Met) to amino acid number 732 (Val); and (i) the amino acidsequence as shown in SEQ ID NO:46 from amino acid number 1 (Met) toamino acid number 649 (Ile).
 2. An isolated polynucleotide comprising asequence selected from the group consisting of: (a) a polynucleotide asshown in SEQ ID NO:1 from nucleotide number 228 to amino acid number851; (b) a polynucleotide as shown in SEQ ID NO:1 from nucleotide number228 to amino acid number 1727; (c) a polynucleotide as shown in SEQ IDNO:1 from nucleotide number 228 to amino acid number 1799; (d) apolynucleotide as shown in SEQ ID NO:1 from nucleotide number 1800 toamino acid number 2366; (e) a polynucleotide as shown in SEQ ID NO:45from nucleotide number 1791 to amino acid number 2108; (f) apolynucleotide as shown in SEQ ID NO:1 from nucleotide number 228 toamino acid number 2366; (g) a polynucleotide as shown in SEQ ID NO:45from nucleotide number 219 to amino acid number 2108; (h) apolynucleotide as shown in SEQ ID NO:1 from nucleotide number 171 toamino acid number 2366; (i) a polynucleotide as shown in SEQ ID NO:45from nucleotide number 162 to amino acid number 2108; and (j) apolynucleotide sequence complementary to (a) through (i).
 3. An isolatedpolynucleotide according to claim 1, wherein the polypeptide furthercomprises a transmembrane domain consisting of residues 520 (Ile) to 543(Leu) of SEQ ID NO:2.
 4. An isolated polynucleotide according to claim1, wherein the polypeptide further comprises an intracellular domainconsisting of residues 544 (Lys) to 732 (Val) of SEQ ID NO:2 or 544(Lys) to 649 (Ile) of SEQ ID NO:46.
 5. An isolated polynucleotideaccording to claim 1, wherein the polypeptide encoded by thepolynucleotide has activity as measured by cell proliferation,activation of transcription of a reporter gene, or wherein thepolypeptide encoded by the polynucleotide further binds to an antibody,wherein the antibody is raised to a polypeptide comprising a sequence ofamino acids from the group consisting of: (a) the polypeptide comprisingamino acid number 20 (Ala) to 227 (Pro) of SEQ ID NO:2; (b) thepolypeptide comprising amino acid number 20 (Ala) to 519 (Glu) of SEQ IDNO:2; (c) the polypeptide comprising amino acid number 20 (Ala) to 543(Leu) of SEQ ID NO:2; (d) the polypeptide comprising amino acid number544 (Lys) to 732 (Val) of SEQ ID NO:2; (e) the polypeptide comprisingamino acid number 544 (Lys) to 649 (Ile) of SEQ ID NO:46; (f) thepolypeptide comprising amino acid number 20 (Ala) to 732 (Val) of SEQ IDNO:2; (g) the polypeptide comprising amino acid number 20 (Ala) to 649(Ile) of SEQ ID NO:46; (h) the polypeptide comprising amino acid number1 (Met) to 732 (Val) of SEQ ID NO:2; and (i) the polypeptide comprisingamino acid number 1 (Met) to 649 (Ile) of SEQ ID NO:46, and wherein thebinding of the antibody to the isolated polypeptide is measured by abiological or biochemical assay including radioimmunoassay,radioimmuno-precipitation, Western blot, or enzyme-linked immunosorbentassay.
 6. An expression vector comprising the following operably linkedelements: a transcription promoter; a DNA segment encoding a polypeptidecomprising an amino acid sequence as shown in SEQ ID NO:2 from aminoacid number 20 (Ala) to 732 (Val) or an amino acid sequence as shown inSEQ ID NO:46 from amino acid number 20 (Ala) to 649 (Ile); and atranscription terminator, wherein the promoter is operably linked to theDNA segment, and the DNA segment is operably linked to the transcriptionterminator.
 7. An expression vector according to claim 6, furthercomprising a secretory signal sequence operably linked to the DNAsegment.
 8. A cultured cell comprising an expression vector according toclaim 7, wherein the cell expresses a polypeptide encoded by the DNAsegment.
 9. An expression vector according to claim 6, wherein the DNAsegment encodes a polypeptide comprising an amino acid sequence as shownin SEQ ID NO:2 from amino acid number 20 (Ala) to 227 (Pro); or as shownin SEQ ID NO:2 from amino acid number 20 (Ala) to 519 (Glu); and atranscription terminator, wherein the promoter, DNA segment, andterminator are operably linked.
 10. An expression vector according toclaim 9, further comprising a secretory signal sequence operably linkedto the DNA segment.
 11. An expression vector according to claim 9,wherein the polypeptide further comprises a transmembrane domainconsisting of residues 520 (Ile) to 543 (Leu) of SEQ ID NO:2.
 12. Anexpression vector according to claim 9 wherein the polypeptide furthercomprises an intracellular domain consisting of residues 544 (Lys) to732 (Val) of SEQ ID NO:2, or residues 544 (Lys) to 649 (Ile) of SEQ IDNO:46.
 13. A cultured cell into which has been introduced an expressionvector according to claim 9, wherein the cell expresses a solublereceptor polypeptide encoded by the DNA segment.
 14. A DNA constructencoding a fusion protein, the DNA construct comprising: a first DNAsegment encoding a polypeptide comprising a sequence of amino acidresidues selected from the group consisting of: (a) the amino acidsequence of SEQ ID NO:2 from amino acid number 1 (Met), to amino acidnumber 19 (Ala); (b) the amino acid sequence of SEQ ID NO:54 from aminoacid number 1 (Met), to amino acid number 32 (Ala); (c) the amino acidsequence of SEQ ID NO:2 from amino acid number 20 (Ala), to amino acidnumber 227 (Pro); (d) the amino acid sequence of SEQ ID NO:2 from aminoacid number 20 (Ala), to amino acid number 519 (Glu); (e) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 20 (Ala) toamino acid number 543 (Leu); (f) the amino acid sequence as shown in SEQID NO:2 from amino acid number 520 (Ile) to amino acid number 543 (Leu);(g) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 544 (Lys) to amino acid number 732 (Val); (h) the amino acidsequence as shown in SEQ ID NO:46 from amino acid number 544 (Lys) toamino acid number 649 (Ile); (i) the amino acid sequence as shown in SEQID NO:2 from amino acid number 20 (Ala) to amino acid number 732 (Val);and (j) the amino acid sequence as shown in SEQ ID NO:46 from amino acidnumber 20 (Ala) to amino acid number 649 (Ile); and at least one otherDNA segment encoding an additional polypeptide, wherein the first andother DNA segments are connected in-frame; and wherein the first andother DNA segments encode the fusion protein.
 15. An expression vectorcomprising the following operably linked elements: a transcriptionpromoter; a DNA construct encoding a fusion protein according to claim14; and a transcription terminator, wherein the promoter is operablylinked to the DNA construct, and the DNA construct is operably linked tothe transcription terminator.
 16. A cultured cell comprising anexpression vector according to claim 15, wherein the cell expresses apolypeptide encoded by the DNA construct.
 17. A method of producing afusion protein comprising: culturing a cell according to claim 16; andisolating the polypeptide produced by the cell.
 18. An isolatedpolypeptide comprising a sequence of amino acid residues selected fromthe group consisting of: (a) the amino acid sequence as shown in SEQ IDNO:2 from amino acid number 20 (Ala) to amino acid number 227 (Pro); (b)the amino acid sequence as shown in SEQ ID NO:2 from amino acid number20 (Ala) to amino acid number 519 (Glu); (c) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 20 (Ala) to amino acidnumber 543 (Leu); (d) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 544 (Lys) to amino acid number 732 (Val); (e) theamino acid sequence as shown in SEQ ID NO:46 from amino acid number 544(Lys) to amino acid number 649 (Ile); (f) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 20 (Ala) to amino acidnumber 732 (Val); (g) the amino acid sequence as shown in SEQ ID NO:46from amino acid number 20 (Ala) to amino acid number 649 (Ile); (h) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 1(Met) to amino acid number 732 (Val); and (i) the amino acid sequence asshown in SEQ ID NO:46 from amino acid number 1 (Met) to amino acidnumber 649 (Ile).
 19. An isolated polypeptide according to claim 18,wherein the polypeptide further comprises a transmembrane domainconsisting of residues 520 (Ile) to 543 (Leu) of SEQ ID NO:2.
 20. Anisolated polypeptide according to claim 18 wherein the polypeptidefurther comprises an intracellular domain consisting of residues 544(Lys) to 732 (Val) of SEQ ID NO:2 or 544 (Lys) to 649 (Ile) of SEQ IDNO:46.
 21. An isolated polynucleotide according to claim 18 wherein thepolypeptide has activity as measured by cell proliferation, activationof transcription of a reporter gene, or wherein the polypeptide encodedby the polynucleotide further binds to an antibody, wherein the antibodyis raised to a polypeptide comprising a sequence of amino acids from thegroup consisting of: (a) the polypeptide comprising amino acid number 20(Ala) to 227 (Pro) of SEQ ID NO:2; (b) the polypeptide comprising aminoacid number 20 (Ala) to 519 (Glu) of SEQ ID NO:2; (c) the polypeptidecomprising amino acid number 20 (Ala) to 543 (Leu) of SEQ ID NO:2; (d)the polypeptide comprising amino acid number 544 (Lys) to 732 (Val) ofSEQ ID NO:2; (e) the polypeptide comprising amino acid number 544 (Lys)to 649 (Ile) of SEQ ID NO:46; (f) the polypeptide comprising amino acidnumber 20 (Ala) to 732 (Val) of SEQ ID NO:2; (g) the polypeptidecomprising amino acid number 20 (Ala) to 649 (Ile) of SEQ ID NO:46; (h)the polypeptide comprising amino acid number 1 (Met) to 732 (Val) of SEQID NO:2; and (i) the polypeptide comprising amino acid number 1 (Met) to649 (Ile) of SEQ ID NO:46, and wherein the binding of the antibody tothe isolated polypeptide is measured by a biological or biochemicalassay including radioimmunoassay, radioimmuno-precipitation, Westernblot, or enzyme-linked immunosorbent assay.
 22. A method of producing apolypeptide comprising: culturing a cell according to claim 8; andisolating the polypeptide produced by the cell.
 23. An isolatedpolypeptide comprising an amino acid segment selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 fromamino acid number 20 (Ala) to amino acid number 227 (Pro); (b) the aminoacid sequence as shown in SEQ ID NO:2 from amino acid number 20 (Ala) toamino acid number 519 (Glu); (c) the amino acid sequence as shown in SEQID NO:18; and (d) the amino acid sequence as shown in SEQ ID NO:22,wherein the polypeptide is substantially free of transmembrane andintracellular domains ordinarily associated with hematopoieticreceptors.
 24. A method of producing a polypeptide comprising: culturinga cell according to claim 13; and isolating the polypeptide produced bythe cell.
 25. A method of producing an antibody to a polypeptidecomprising: inoculating an animal with a polypeptide selected from thegroup consisting of: (a) a polypeptide consisting of 9 to 713 aminoacids, wherein the polypeptide comprises a contiguous sequence of aminoacids in SEQ ID NO:2 from amino acid number 20 (Ala), to amino acidnumber 732 (Val); (b) a polypeptide consisting of 9 to 630 amino acids,wherein the polypeptide comprises a contiguous sequence of amino acidsin SEQ ID NO:46 from amino acid number 20 (Ala), to amino acid number649 (Ile); (c) a polypeptide comprising amino acid number 20 (Ala) to227 (Pro) of SEQ ID NO:2; (d) a polypeptide comprising amino acid number20 (Ala) to 519 (Glu) of SEQ ID NO:2; (e) a polypeptide comprising aminoacid number 20 (Ala) to 543 (Leu) of SEQ ID NO:2; (f) a polypeptidecomprising amino acid number 544 (Lys) to 732 (Val) of SEQ ID NO:2; (g)a polypeptide comprising amino acid number 544 (Lys) to 649 (Ile) of SEQID NO:46; (h) a polypeptide comprising amino acid number 20 (Ala) to 732(Val) of SEQ ID NO:2; (i) a polypeptide comprising amino acid number 20(Ala) to 649 (Ile) of SEQ ID NO:46; (j) a polypeptide comprising aminoacid number 1 (Met) to 732 (Val) of SEQ ID NO:2; (k) a polypeptidecomprising amino acid number 1 (Met) to 649 (Ile) of SEQ ID NO:46, (l) apolypeptide comprising amino acid residues 43 through 48 of SEQ ID NO:2;(m) a polypeptide comprising amino acid residues 157 through 162 of SEQID NO:2; (n) a polypeptide comprising amino acid residues 158 through163 of SEQ ID NO:2; (o) a polypeptide comprising amino acid residues 221through 226 of SEQ ID NO:2; and (p) a polypeptide comprising amino acidresidues 426 through 431 of SEQ ID NO:2; and wherein the polypeptideelicits an immune response in the animal to produce the antibody; andisolating the antibody from the animal.
 26. An antibody produced by themethod of claim 25, which specifically binds to a polypeptide of SEQ IDNO:2 or SEQ ID NO:46.
 27. The antibody of claim 26, wherein the antibodyis a monoclonal antibody.
 28. An antibody that specifically binds to apolypeptide of claim
 18. 29. A method of detecting, in a test sample,the presence of a modulator of cytokine receptor protein activity,comprising: culturing a cell into which has been introduced anexpression vector according to claim 9, wherein the cell expresses theprotein encoded by the DNA segment in the presence and absence of a testsample; and comparing levels of activity of the protein in the presenceand absence of a test sample, by a biological or biochemical assay; anddetermining from the comparison, the presence of modulator of theprotein activity in the test sample.
 30. A method for detecting acytokine receptor ligand within a test sample, comprising: contacting atest sample with a polypeptide comprising an amino acid sequence fromthe group consisting of: (a) the amino acid sequence as shown in SEQ IDNO:2 from amino acid number 20 (Ala) to amino acid number 227 (Pro); (b)the amino acid sequence as shown in SEQ ID NO:2 from amino acid number20 (Ala) to amino acid number 519 (Glu); (e) the amino acid sequence asshown in SEQ ID NO:18; the amino acid sequence as shown in SEQ ID NO:22;and detecting the binding of the polypeptide to a ligand in the sample.31. A method according to claim 30 wherein the polypeptide is membranebound within a cultured cell, and the detecting step comprises measuringa biological response in the cultured cell.
 32. A method according toclaim 31 wherein the biological response is cell proliferation oractivation of transcription of a reporter gene.